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BwoB iGEM 2019 · Benchling

BwoB iGEM 2019

Project: 2019 iGEM

Authors: Ellen Jorgensen

Friday, 5/24/19

Mentor: Craig

PI: Ellen

Administrator: Chris

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Introductions of members

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Biobuilder: download Riley

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iGEM Academy on Youtube

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Collection of videos: parodies of molecular biology

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Helpful for a basis

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Introduce iGEM competition description to BEAM center students

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Restriction Digest

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2017: targeted psyllid (vector) so it’d have trouble carrying virus

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Talking Biotech Podcast 185 by Dr. Steven Savage

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Reach out about citrus virus -- use Tristeza virus

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Tristeza: 1960s virus mutated inside citrus plant

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DNA Learning Center: educational videos of crystallography

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Intro to pipette usage -- hands-on practice

Friday, 5/31/19

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Watched video on recombinant DNA (dnalc-mechanisms of recombination)

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Cut plasmids with restriction enzymes

Friday, 6/7/19

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Showed video to introduce restriction enzyme

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Displayed process for agarose gel for electrophoresis (1% gel = 0.5 TAE Solution)

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Demonstrated how to prep gel, pour it, and add ethidium bromide

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Restriction digest products still in the fridge

Friday, 6/14/19

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Introduced Google Classroom as main communication platform

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Google Hangout & Discord when unable to attend in person -- useful video broadcast over channels

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Four interns in lab to mentor students

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Safety lab forms: for iGEM and Biotech Without Borders

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Review Restriction Digest: enzymes = Bozeman Science video

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Polymerase Chain Reaction (PCR)

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Walk through and introduced basic lab

Friday, 6/21/19

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Safety agreements & forms (1): If under 18, need signatures of parents

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Classroom link with online version available

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Deliverables on iGEM ~ test: answer questions

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Team wiki (see 2017 fancy team wiki), team poster, team presentation, judging form, project attributors, safety, registry part pages, project inspiration

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iGEM account: igem.org → fill out personal info as prompted → login to account → membership code to team: 327718

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Pending queue -- need verification

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Keep up with Google Classroom account (3); Biotech Without Borders (4) forms

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Video call with Dr. Georgios Vidalakis of the University of California, Riverside

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1998-2005: FL neglected severity of HLB insect - disease spread and threatens the citrus industry; FL has lost half the industry; 2008: CA industries jeopardized

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Improve RATE of detection of bacteria in leaves; create a quiz that allows kids across the country to help diagnose millions of leaves (virtual game); trap the psyllids with traps (acetic acid); infrared cameras track disease

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Localized immunity issues? signals are systemic; surrogate bacteria in mango (cultures that can be sent over) ALSO can send over actual bacteria Diaphorina citri after arranging the permits; california suspecting peptides & concern with consumers; smaller virus RNA -> easier to work with the tristeza virus

○

https://news.ucr.edu/articles/2019/04/01/plant-pathologist-leads-research-stop-spread-citrus-destroying-disease

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See Craig for head start with lab work → increases expertise & familiarity

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Start with scheduling with students -- genetic engineering tasks

Friday, 6/28/19

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Established that the actual bacteria Candidatus Liberibacter asiaticus would not be easily culturable

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Considered periwinkle as a cultured plant & discussed the surrogate bacteria as usable: Liberibacter crescens

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Reviewed restriction enzyme & bioengineering techniques with our team members

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Established that we will create a written protocol for new techniques for members away at Guatemala

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Introduced t-shirt design project & team project names

Monday, 7/8/19

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Practice lab techniques

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Pipet

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Ligation

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Colony PCR

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Gel Electrophoresis

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Transformation

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Restriction Digest

Tuesday, 7/9/19

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PCR Polymerase Chain Reaction

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Amplify a certain area of DNA

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1. Break Apart Double Helix (Heat it up)- Denaturing

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2. Primers sit on complementary base pair

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3. Extend the areas after primers

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1 X TAE contents:

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C₁V₁=C₂V₂ (C is concentration, V is volume)

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Gel Electrophoresis:

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1. Pour out 50 mL 1 X TAE into flask

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2. Add .35 g agarose

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3. Cover Flask with Plastic Wrap

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4. Put in microwave for 1 minute 15 seconds (check every 30 seconds)

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5. Pour into gel mold and add 5 microliters of ethidium bromide

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6. Put in combs and let sit for 30 minutes

Friday, 7/12/19

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Remove syllids as they pick up RNAi + cloning project

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Fluorescent protein genes picked out

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iGEM BioBrick

iGEM_Meeting_Notebook.pdf

Tuesday, 7/16/19

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PCR Digestion (Restriction DIgest)

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1 mL DNA

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5 mL NEB Buffer 2.1

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1 ml EcoR1 HF

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1 mL Pst 1

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50 mL MGBW

Thursday, 7/18/19

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DIstribution Kit 2019

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Transformation:

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2 types of transformations: Electric Shock and Heat Shock

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Wash cell in buffer solution to weaken the cell walls

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Mini Prep: (Mini Prep kit + Monarch)

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Spin colony to get the pellet on the bottom. Pellet 1-5 mL

Monday, 7/22/19

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Overnight Prep For Mini Prep (Grow Colonies)

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5000 microliters of LB

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5 microliters Chlor

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10 microliters colony PCR

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1. Mix everything together

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2. Parafilm the tube

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3. Put in the Rotissirie

Thursday, 7/25/19

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Transformation for Red Colonies

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Plasmid RFP + AMP

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DNA Distribution Kit

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Linearized back bones, competent cell test kit, measurement kit

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Reporter quantitizes how bright the colors are

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Insert conatins ribosome, binding site

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Bacterial Transformation: Method of foreign DNA being introduced into competent cells. Bacteria takes up the plasmids and expresses the specific gene in the DNA

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Colony PCR:

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Inside Thermocycler:

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Denaturation- separation of double stranded DNA

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Annealing- laying of primers on template DNA

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Extension- polymerase synthesizes new strands of DNA using dNTP's

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Check product by running a .7% gel

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Combine colonies with LB broth and antibiotics

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Rotate overnight

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Mini prep

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Method of extracting and purifying the plasmid DNA from bacterial cells. Results in large source of DNA that is pure and contains desired construction in a circulated form used for bacterial transformation or PCR reaction

Tuesday, 7/30/19

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Another transformation

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Overnight prep (we want more bacteria with the plasmid)

Wednesday, 7/31/19

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Transcription of DNA in bacteria

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DNA to RNA to Protien

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Mini Prep (followed given instructions)

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Nanodropped the concentrations made

Thursday, 8/1/19

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Transformation (GFP + AMP) and Gel Electrophorises

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RNAi

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Used to control genes

Monday, 8/12/19

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Transformed Ecoli cells with Gblock+Chlor plasmids

Tuesday, 8/13/19

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Looked at plates from transformation on 8/12

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We had a few colonies that succesfully transformed

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Took colonies from each plate and suspended them in seperate tubes with 20 microliters of MBGW (template)

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Tubes were labeled S1, S2, & S3, corresponding to plates that colonies were sourced from

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Performed PCR with 1 micro templates from

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12.5 microliters of Q5 Master Mix

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1.5 microliters of VR Primer

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1.5 microliters of VF2 Primer

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1 microliter of the template

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8.5 microliters of MBGW

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Loaded test tubest with Colony PCR reagents into the Thermocycler

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Cast a 0.7% Agarose gel to perform Gel electrophoresis with products from our PCR

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Replenished 1x working stock of TAE from 50x stock

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Performed C₁V₁ = C₂V₂

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(50) V₁ = (1) 1000 mL

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V₁ = 20

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Added 20 mL of 50x TAE stock into 980 mL of dH2O to create 1000 mL of 1x TAE working stock

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Mixed PCR products with gel

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Ran gel

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No bands

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Was most likely due to the fact that we did not spin down the the PCR reagents in their tubes prior to loading into thermocycler

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Will Evaluate tonight

Wednesday, 8/14/19

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Performed mini preparation of plasmid DNA: GFP + C, RFP + A

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Repeatedly centrifuged and added wash buffers to DNA in order to isolate plasmid DNA from bacteria

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Known as alkaline lysis

Thursday, 8/15/19

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Made and poured plates

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Mixed 8.75 grams of agar with 250 milliters of distilled water

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Put the mixture in a pressure cooker for 30 minutes

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Let the mixture cool down for ten minutes

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Add antibiotic to the mixture

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Pour mixture onto plates

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Performed a transformation using Ecoli T7 Express cells

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Performed transformation on GFP + C made in mini prep on 8/14 to make sure we labled the tubes correctly

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Constitutive promoter: always displaying trait, inducible promoters, repressible promoters

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Initiation - Elongation - Termination (independent hairpin loop forms)

Monday, 8/19/19

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We found out that while ligating out backbone folded onto itself. This prevented out insert from ligating with the backbone to form a full plasmid. To troubleshoot this we decided to use an enzyme called "Antartic Phosphatase" to cut the ends of the backbone. This will dephophate the backbone and prevent it from folding onto itself.

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Dephosphating Backbone - 2microliter buffer, 1 microliter ant(Antartic Phosphatase), 10 microliter backbone, 7 mucroliter mgbw

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Ligation- 2 microliter buffer, 1 microliter dephosphated DNA, 1 microliter ligase, 16 microliter insert

Tuesday, 8/20/19

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We are transforming out newly made DNA from 8/19. If out dephophatase worked then the cells should grow with a blue flourescent color.

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Take out E-Coli(c2987) cells and defrost them from nitrogen.

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Put 4 chlor pretri dishes in an incubator and make an ice bath.

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Pipette 50uL of E-Coli and put them into tubeG-Block 1, G-Block 2, G-Block 3, and RFP control.

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Pipette our designated plasmids onto each of our tube. 3uL for G-Block, 1uL for RFP.

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Heat shock the tubes for 45secs at 42 ddegrees.

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Take them out and put them in icebath for 10 min.

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Add 950 uL of LB to the tubes.

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Put parafilm on the tube and place in rotisserie for a hour.

Tuesday, 8/27/19

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Pasmid Prep (Mini Prep) for blue chromo protein

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Gel electrophoresis from the result of the plasmid prep

Tuesday, 9/3/19

Workflow for the day was to inoculate colonies from separate plasmid cassette transformation into overnight culture, that will be used to create individual plasmid preps for Wednesday 9/4.

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First we counted all the colonies on the petre dishes, and got tubes for each colony (24).

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Next we placed the 3 millimeters of Lb +Chlor.

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Then we scraped each of the colonies off of the petre dishes using the loops and placed them into the tubes of Lb + Chlor.

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Then we used film to seal Oof the tubes and placed them into the shaker incubator to grow up overnight.

Wednesday, 9/4/19

Plasmid Prep

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take out 24 large tubes of Lb + Chlor AK and SOB

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Pipette 2 microliter for each of the Lb + Chlor AK and SOB into the microcentrifuge tubes

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place them balanced and spaced eqally in centrifugation for 30 seconds

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Discard by pipetting liquid out and leave the cells at the bottom

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pipette 2 microliter of Lb + Chlor back inside the tube. Then repeat the process of placing in the centrifugation for 30 seconds

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Pipette 200 microliter of Plasmid Resuspension Buffer into the tube and slowly break or mix with the cells. (until the solid is gone)

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Add 200 microliter of Plasmid Lysis Buffer in the tube and shake it for 5 times. Set for 1 minute at a room temperature.

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Add 400 microliter of Plasmid Neutralization Buffer in the tube and shake it until the color completely changes. Set for 2 minutes at room temperatur

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Back to centrifugation (balanced and spaced equally) for 5 minutes.

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Transfer the supernatant to the plasmid column and place it into the centrifugation for 4 minutes.

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Transfer the column into a new tube and add 200 microliter of Plamid Wash Buffer 1. Then place it into the centrifugation for 1 minute.

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Add 400 microliter of Plasmid Wash Buffer 2 into the column and place it into the centriguation for 1 minute.

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Transfer the columns to a new microfuge tube and add 50 microliters of DNA Elution Buffer in the tube. Wait for 1 minute and place it into the centrifugation for 1 minute.

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Separate the columns. The leftover liquid is the DNA sample.

Thursday, 9/5/19

Plasmid Prep through Gel

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Put the weigh paper on a gram scale. Then measure 0.35 g of agarose.

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50mL of 1X TAE and put it into a erlenmeyer flask. Put the 0.35 g of agarose in the flask and slowly swirl / mix it.

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cover the top of the elenmeyer flask with a clean wrap foil and poke the top.

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Put it into a microwave for 30 seconds.

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Then slowly swirl it and put it in the microwave again for 30 seconds.

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Then slowly swril it and put it in the microwave for 15 seconds. Cool it after it's done.

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Prepare the Gel electrophoresis / Gel box. Then pour the agarose liquid into the Gel box and cool it for a while.

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After cooling, put 5 microliter of Bromide in the gel liquid and swirl / mix it.

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Place the comb and make the wells. Wait for 30 minutes.

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Replace the Gel and put a black piece of paper under the gel box for better observations.

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pour the 1X TAE and fill the gel box until it covers the gel.

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prepare the parafilms, ladder, purple dye, and the AK and SOB DNA samples.

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Then pipette 6 microliter of ladder and slowly + gently place it in one of the wells.

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Then pipette 1 microliter of purple dye and place it on the parafilm.

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add 5 microliter of one of the DNA samples and mix it with the purple dye.

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Then pipette as 6 microliter of the mixed purple dye and DNA sample and place it to the next well of the ladder.

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*repeat the process for each of the DNA samples.

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After filling the wells, carry it to the power source and set as 80V. Put the cover on top of the gel box and set it as 'run' for 65minutes.

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After 65 minutes, take the gel out and pour out the liquid.

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Put the gel in the UV transilluminator and turn the power on.

Sequential Double Digest

Introduction

This is the Sequential Double Digest Protocol with Standard Restriction Enzymes. If there is no buffer in which the two enzymes exhibit > 50% activity, this sequential digest can be performed. More information from NEB can be found here. Double Digests can be designed using NEB's Double Digest Finder. NEBcloner will help guide your reaction buffer selection when setting up double digests. See the NEBuffer Activity/Performance Chart with Restriction Enzymes for the incubation temperatures.

Materials

DNA 1 µg
NEBuffer
- 1X
NEB Restriction Enzymes
Deionized Water

Procedure

Sequential Double Digest

Set up the following reaction using the restriction endonuclease that has the lowest salt concentration in its recommended buffer (total reaction volume 50 µl).

Set up the following digest reaction on ice

	A	B	C
1		Volume (µl)
2	Buffer (10x)	5
3	DNA *	Input Volume for ng
4	Restriction Enzyme #1 **	1
5	Deionized Water (μl)	1
6	Total Volume (μl)	#VALUE!

Table1

*A 50 µl reaction volume is recommended for digestion of 1 µg of substrate.

** Restriction Enzyme, 10 units is sufficient, generally 1 µl is used

***The enzyme should be the last component added to reaction

Mix components by pipetting the reaction mixture up and down, or by "flicking" the reaction tube.

Quick ("touch") spin-down in a microcentrifuge. Do not vortex the reaction.

Incubate for 1 hour at the enzyme-specific appropriate temperature.

01:00:00

Can be decreased to 5-15 minutes by using a Time-Saver™ Qualified Restriction Enzyme

See the NEBuffer Activity/Performance Chart with Restriction Enzymes for the incubation temperatures.

Adjust the salt concentration of the reaction (using a small volume of a concentrated salt solution) to approximate the reaction conditions of the second restriction endonuclease.

Alternatively, a spin column can be used to isolate the DNA prior to the second reaction.

Add the second enzyme.

** Restriction Enzyme #2, 10 units is sufficient, generally 1 µl is used

Mix components by pipetting the reaction mixture up and down, or by "flicking" the reaction tube.

Quick ("touch") spin-down in a microcentrifuge. Do not vortex the reaction.

Incubate for 1 hour at the enzyme-specific appropriate temperature.

Can be decreased to 5-15 minutes by using a Time-Saver™ Qualified Restriction Enzyme

See the NEBuffer Activity/Performance Chart with Restriction Enzymes for the incubation temperatures.

Ligation Protocol WITH T4 DNA Ligase (M0202)

Introduction

Please see the NEB website for supporting information on this protocol.

Materials

10X T4 DNA Ligase Reaction Buffer
T4 DNA Ligase
Vector DNA (4kb)
Insert DNA (1kb)
Nuclease-free water

Procedure

Set up the T4 DNA Ligase Reaction

Note: T4 DNA Ligase should be added last. The table shows a ligation using a molar ratio of 1:3 vector to insert for the indicated DNA sizes. Use NEB calculator to calculate molar ratios.

Thaw the T4 DNA Ligase Buffer and resuspend at room temperature.

Tip: Alicuote the 10x buffer less concentrated so when thawing, the DTT gets soluble more easily.

Set up the following reaction in a microcentrifuge tube on ice:

	A	B
1	Component	Volume (μl)
2	10X T4 DNA Ligase Buffer	2
3	Vector DNA: 50 ng (0.020 pmol)
4	Insert DNA: 37.5 ng (0.060 pmol)
5	Nuclease-free water	17
6	T4 DNA Ligase	1
7	Total	20

Table1

Gently mix the reaction by pipetting up and down and microfuge briefly.

For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes. For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours.

Heat inactivate at 65 degrees C for 10 minutes.

00:10:00

Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.

Use 25 uL DH5α cells, and add 2 uL of reaction mixture.

Transformation

Introduction

A process in which we stress competent cells in a series of times heat shocks and ice baths. This allows the cells to take in the plasmids we want them to have.

Materials

100 μL aliquots of competent cells
ice block
DNA ligation mix
micropipettes and sterile pipette tips
Glass beads
control plasmid DNA
water bath at 42 °C (or 37 °C)
Luria Broth
Incubator 37 °C with a rotisserie
Luria Broth agar plates (with desired antibiotic)
Heatblock

Procedure

Transformation Protocol

Obtain competent cells from a -80°C freezer and let it thaw on ice for 5 minutes .

After labeling each tube, use a sterile tip to add 5 μL of the ligation mix to the competent cells and leave them on ice for one hour.

Using a Heatblock for 45 sec. at 42 degrees C. (Remember to turn off the heatblock.)

Immediately after heat schocking the cells, place them back on ice for 5 minutes.

Using a sterile tip add 950 μL Luria broth to the cells and parafilm the tubes

Place the parafilmed tubes into a rotisserie for 1 hour.

Once the tubes are done incubating take off the parafilm.

Warm the Luria broth agar plates with the desired antibiotic resitstance at room temperature.

Label the plates with the desired product, name, and date.

Plate 100 μL of the transformed cells in to seperate plates.

Place 3 to 5 glass beads into the plate to spread the cells evenly.

Shake the plates in an up and down motion for 2 minutes.

Remove beads and dispose of them into a waste bin.

Parafilm the plates and place in the rotisserie at 37°C for 16 hours.

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class="mediocre-item is-text" data-scroll-id="note-part916"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part916" data-index="4" style="margin-left: 35px;">Introductions of members </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part917"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part917" data-index="5" style="margin-left: 35px;">Biobuilder: download Riley </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part918"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part918" data-index="6" style="margin-left: 35px;">iGEM Academy on Youtube </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part919"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part919" data-index="7" style="margin-left: 70px;">Collection of videos: parodies of molecular biology </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part920"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part920" data-index="8" style="margin-left: 70px;">Helpful for a basis </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part921"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part921" data-index="9" style="margin-left: 35px;">Introduce iGEM competition description to BEAM center students </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part922"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part922" data-index="10" style="margin-left: 35px;">Restriction Digest </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part923"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part923" data-index="11" style="margin-left: 70px;"><span style="color:#344043"><span style="background-color:#FFFFFF">2017: targeted psyllid (vector) so it’d have trouble carrying virus</span></span><span style="color:#344043"><span style="background-color:#FFFFFF"> </span></span></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part924"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part924" data-index="12" style="margin-left: 70px;"><span style="color:#344043"><span style="background-color:#FFFFFF">Talking Biotech Podcast 185 by Dr. Steven Savage</span></span></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part925"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 105px;"><span>■</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part925" data-index="13" style="margin-left: 105px;"><span style="color:#344043"><span style="background-color:#FFFFFF">Reach out about citrus virus -- use Tristeza virus</span></span></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part926"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part926" data-index="14" style="margin-left: 70px;"><span style="color:#344043"><span style="background-color:#FFFFFF">Tristeza: 1960s virus mutated inside citrus plant</span></span></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part927"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part927" data-index="15" style="margin-left: 35px;"><span style="color:#344043"><span style="background-color:#FFFFFF">DNA Learning Center: educational videos of crystallography</span></span></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part928"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part928" data-index="16" style="margin-left: 35px;"><span style="color:#344043"><span style="background-color:#FFFFFF">Intro to pipette usage -- hands-on practice</span></span></div></div></div><div class="daySeparator" data-scroll-id="entryDay860"><div class="daySeparator-inputWrapper"><input class="daySeparator-inputWrapper-hiddenInput" type="input"/></div><span class="daySeparator-date">Friday, 5/31/19</span><div class="daySeparator-line"></div></div><div class="mediocre-item is-text" data-scroll-id="note-part929"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part929" data-index="17" style="margin-left: 35px;"> Watched video on recombinant DNA (dnalc-mechanisms of recombination)</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part930"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part930" data-index="18" style="margin-left: 35px;"> Cut plasmids with restriction enzymes</div></div></div><div class="daySeparator" data-scroll-id="entryDay862"><div class="daySeparator-inputWrapper"><input class="daySeparator-inputWrapper-hiddenInput" type="input"/></div><span class="daySeparator-date">Friday, 6/7/19</span><div class="daySeparator-line"></div></div><div class="mediocre-item is-text" data-scroll-id="note-part931"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part931" data-index="19" style="margin-left: 35px;">Showed video to introduce restriction enzyme</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part932"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part932" data-index="20" style="margin-left: 35px;">Displayed process for agarose gel for electrophoresis (1% gel = 0.5 TAE Solution)</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part933"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part933" data-index="21" style="margin-left: 35px;">Demonstrated how to prep gel, pour it, and add ethidium bromide</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part934"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part934" data-index="22" style="margin-left: 70px;">Restriction digest products still in the fridge </div></div></div><div class="daySeparator" data-scroll-id="entryDay864"><div class="daySeparator-inputWrapper"><input class="daySeparator-inputWrapper-hiddenInput" type="input"/></div><span class="daySeparator-date">Friday, 6/14/19</span><div class="daySeparator-line"></div></div><div class="mediocre-item is-text" data-scroll-id="note-part935"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part935" data-index="23" style="margin-left: 35px;"><span style="color:#000000">Introduced Google Classroom as main communication platform</span></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part936"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part936" data-index="24" style="margin-left: 70px;"><span style="color:#000000">Google Hangout </span><span style="color:#000000">&amp; </span><span style="color:#000000">Discord </span><span style="color:#000000">when unable to attend in person -- useful video broadcast over channels</span></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part937"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part937" data-index="25" style="margin-left: 70px;"><span style="color:#000000">Four interns in lab to mentor students</span></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part938"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part938" data-index="26" style="margin-left: 35px;"><span style="color:#000000">Safety lab forms: for iGEM and Biotech Without Borders</span></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part939"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part939" data-index="27" style="margin-left: 35px;"><span style="color:#000000">Review </span><span style="color:#000000">Restriction Digest</span><span style="color:#000000">: enzymes = Bozeman Science video</span></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part940"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part940" data-index="28" style="margin-left: 35px;"><span style="color:#000000">Polymerase Chain Reaction (PCR)</span></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part941"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part941" data-index="29" style="margin-left: 35px;"><span style="color:#000000">Walk through and introduced basic lab</span></div></div></div><div class="daySeparator" data-scroll-id="entryDay866"><div class="daySeparator-inputWrapper"><input class="daySeparator-inputWrapper-hiddenInput" type="input"/></div><span class="daySeparator-date">Friday, 6/21/19</span><div class="daySeparator-line"></div></div><div class="mediocre-item is-text" data-scroll-id="note-part942"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part942" data-index="30" style="margin-left: 35px;"><span style="color:#000000">Safety agreements &amp; forms (1)</span><span style="color:#000000">:</span><span style="color:#000000"> </span><span style="color:#000000">If under 18, need signatures of parents</span></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part943"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part943" data-index="31" style="margin-left: 70px;"><span style="color:#000000">Classroom link with online version available</span></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part944"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part944" data-index="32" style="margin-left: 35px;"><i><span style="color:#000000">Deliverables</span></i><span style="color:#000000"> on iGEM ~ test: answer </span><i><span style="color:#000000">questions</span></i></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part945"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part945" data-index="33" style="margin-left: 70px;"><span style="color:#000000">Team wiki (see 2017 fancy team wiki), team poster, team presentation, judging form, project attributors, safety, registry part pages, project inspiration</span></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part946"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part946" data-index="34" style="margin-left: 35px;"><span style="color:#000000">iGEM account</span><span style="color:#000000">: igem.org → fill out personal info as prompted → login to account → membership code to team: 327718</span></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part947"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part947" data-index="35" style="margin-left: 70px;"><span style="color:#000000">Pending queue -- need verification</span></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part948"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part948" data-index="36" style="margin-left: 35px;"><span style="color:#000000">Keep up with </span><span style="color:#000000">Google Classroom account (3)</span><span style="color:#000000">; </span><span style="color:#000000">Biotech Without Borders (4)</span><span style="color:#000000"> forms</span></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part949"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part949" data-index="37" style="margin-left: 35px;"><span style="color:#000000">Video call with </span><span style="color:#000000">Dr. Georgios Vidalakis</span><span style="color:#000000"> of the University of California, Riverside</span></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part950"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part950" data-index="38" style="margin-left: 70px;"><span style="color:#000000">1998-2005: FL neglected severity of HLB insect - disease spread and threatens the citrus industry; FL has lost half the industry; 2008: CA industries jeopardized</span></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part951"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part951" data-index="39" style="margin-left: 70px;"><span style="color:#000000">Improve RATE of detection of bacteria in leaves; create a quiz that allows kids across the country to help diagnose millions of leaves (virtual game); trap the psyllids with traps (acetic acid); infrared cameras track disease</span></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part952"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part952" data-index="40" style="margin-left: 70px;"><span style="color:#000000">Localized immunity issues? signals are systemic; surrogate bacteria in mango (cultures that can be sent over) ALSO can send over actual bacteria </span><u><span style="color:#000000"><span style="background-color:#FFFFFF">Diaphorina citri</span></span></u><span style="color:#000000"> </span><span style="color:#000000">after arranging the permits; california suspecting peptides &amp; concern with consumers; smaller virus RNA -&gt; easier to work with the tristeza virus</span></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part953"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part953" data-index="41" style="margin-left: 70px;"><a href="https://news.ucr.edu/articles/2019/04/01/plant-pathologist-leads-research-stop-spread-citrus-destroying-disease" rel="nofollow noopener noreferrer" target="_blank"><u><span style="color:#1155CC">https://news.ucr.edu/articles/2019/04/01/plant-pathologist-leads-research-stop-spread-citrus-destroying-disease</span></u></a></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part954"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part954" data-index="42" style="margin-left: 35px;"><span style="color:#000000">See Craig for head start with lab work → increases expertise &amp; familiarity</span></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part955"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part955" data-index="43" style="margin-left: 70px;"><span style="color:#000000">Start with scheduling with students -- genetic engineering tasks</span></div></div></div><div class="daySeparator" data-scroll-id="entryDay868"><div class="daySeparator-inputWrapper"><input class="daySeparator-inputWrapper-hiddenInput" type="input"/></div><span class="daySeparator-date">Friday, 6/28/19</span><div class="daySeparator-line"></div></div><div class="mediocre-item is-text" data-scroll-id="note-part956"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part956" data-index="44" style="margin-left: 35px;"><span style="color:#222222"><span style="background-color:#FFFFFF">Established that the </span></span><span style="color:#222222"><span style="background-color:#FFFFFF">actual bacteria </span></span><i><span style="color:#222222"><span style="background-color:#FFFFFF">Candidatus Liberibacter asiaticus</span></span></i><span style="color:#222222"><span style="background-color:#FFFFFF"> would not be easily culturable</span></span></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part957"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part957" data-index="45" style="margin-left: 35px;"><span style="color:#222222"><span style="background-color:#FFFFFF">Considered periwinkle as a cultured plant &amp; discussed the surrogate bacteria as usable: </span></span><i><span style="color:#222222"><span style="background-color:#FFFFFF">Liberibacter crescens</span></span></i></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part958"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part958" data-index="46" style="margin-left: 35px;"><span style="color:#222222"><span style="background-color:#FFFFFF">Reviewed restriction enzyme &amp; bioengineering techniques with our team members</span></span></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part959"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part959" data-index="47" style="margin-left: 35px;"><span style="color:#222222"><span style="background-color:#FFFFFF">Established that we will create a written protocol for new techniques for members away at Guatemala</span></span></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part960"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part960" data-index="48" style="margin-left: 35px;"><span style="color:#222222"><span style="background-color:#FFFFFF">Introduced t-shirt design project &amp; team project names</span></span></div></div></div><div class="daySeparator" data-scroll-id="entryDay870"><div class="daySeparator-inputWrapper"><input class="daySeparator-inputWrapper-hiddenInput" type="input"/></div><span class="daySeparator-date">Monday, 7/8/19</span><div class="daySeparator-line"></div></div><div class="mediocre-item is-text" data-scroll-id="note-part961"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part961" data-index="49" style="margin-left: 35px;"><span style="color:#000000">Practice lab techniques</span></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part962"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part962" data-index="50" style="margin-left: 70px;"><span style="color:#000000">Pipet</span></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part963"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part963" data-index="51" style="margin-left: 70px;"><span style="color:#000000">Ligation</span></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part964"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part964" data-index="52" style="margin-left: 70px;"><span style="color:#000000">Colony PCR</span></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part965"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part965" data-index="53" style="margin-left: 70px;"><span style="color:#000000">Gel Electrophoresis</span></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part966"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part966" data-index="54" style="margin-left: 70px;"><span style="color:#000000">Transformation</span></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part967"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part967" data-index="55" style="margin-left: 70px;"><span style="color:#000000">Restriction Digest</span></div></div></div><div class="daySeparator" data-scroll-id="entryDay872"><div class="daySeparator-inputWrapper"><input class="daySeparator-inputWrapper-hiddenInput" type="input"/></div><span class="daySeparator-date">Tuesday, 7/9/19</span><div class="daySeparator-line"></div></div><div class="mediocre-item is-text" data-scroll-id="note-part968"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part968" data-index="56" style="margin-left: 35px;">PCR Polymerase Chain Reaction</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part969"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part969" data-index="57" style="margin-left: 70px;">Amplify a certain area of DNA</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part970"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 105px;"><span>■</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part970" data-index="58" style="margin-left: 105px;">1. Break Apart Double Helix (Heat it up)- Denaturing</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part971"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 105px;"><span>■</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part971" data-index="59" style="margin-left: 105px;">2. Primers sit on complementary base pair</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part972"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 105px;"><span>■</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part972" data-index="60" style="margin-left: 105px;">3. Extend the areas after primers</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part973"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part973" data-index="61" style="margin-left: 35px;">1 X TAE contents:</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part974"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part974" data-index="62" style="margin-left: 70px;">C<sub>1</sub>V<sub>1</sub>=C<sub>2</sub>V<sub>2</sub>   (C is concentration, V is volume)</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part975"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part975" data-index="63" style="margin-left: 35px;">Gel Electrophoresis: </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part976"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part976" data-index="64" style="margin-left: 70px;">1. Pour out 50 mL 1 X TAE into flask</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part977"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part977" data-index="65" style="margin-left: 70px;">2. Add .35 g agarose</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part978"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part978" data-index="66" style="margin-left: 70px;">3. Cover Flask with Plastic Wrap</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part979"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part979" data-index="67" style="margin-left: 70px;">4. Put in microwave for 1 minute 15 seconds (check every 30 seconds)</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part980"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part980" data-index="68" style="margin-left: 70px;">5. Pour into gel mold and add 5 microliters of ethidium bromide </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part981"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part981" data-index="69" style="margin-left: 70px;">6. Put in combs and let sit for 30 minutes</div></div></div><div class="daySeparator" data-scroll-id="entryDay874"><div class="daySeparator-inputWrapper"><input class="daySeparator-inputWrapper-hiddenInput" type="input"/></div><span class="daySeparator-date">Friday, 7/12/19</span><div class="daySeparator-line"></div></div><div class="mediocre-item is-text" data-scroll-id="note-part982"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part982" data-index="70" style="margin-left: 35px;"><span style="color:#000000">Remove syllids as they pick up RNAi + cloning project</span></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part983"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part983" data-index="71" style="margin-left: 35px;"><span style="color:#000000">Fluorescent protein genes picked out</span></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part984"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part984" data-index="72" style="margin-left: 35px;"><span style="color:#000000">iGEM BioBrick</span></div></div></div><div class="mediocre-item" data-scroll-id="note-part985"><div class=""><div><div class="hiddenFocusEditable editable" contenteditable="true" data-cid="note-part985-item" data-formattable="true" data-index="73" data-subindex="item"></div><div class="mediocre-captionEditable multi-editable"><div class="mediocre-captionEditable-indent" style="padding-left: 0px;"><div class="mediocre-captionEditable-grid"><div class="mediocre-captionEditable-row"><div class="mediocre-captionEditable-cell mediocre-captionEditable-component"><div class="note-item-wrapper"><div class="dnd-drop-container"><div class="noteLinkedFilePreview mediocre-itemSelect note-item"><div><h5 class="u-no-spacing-top u-no-spacing-bottom"><div class="pull-right"></div><div class="pull-right"><span></span></div><span class="glyphicon glyphicon-paperclip note-icon u-small-spacing-right pull-left" icon="paperclip"></span><div class="note-itemName">iGEM_Meeting_Notebook.pdf</div></h5><div class=""></div></div></div><div class="dnd-drop-container-border"></div></div></div></div><div class="mediocre-captionEditable-cell" style="width: 0px;"></div><div class="mediocre-captionEditable-cell mediocre-captionEditable-caption editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="true" data-cid="note-part985" data-index="73">
+</div></div></div></div></div></div></div></div><div class="daySeparator" data-scroll-id="entryDay876"><div class="daySeparator-inputWrapper"><input class="daySeparator-inputWrapper-hiddenInput" type="input"/></div><span class="daySeparator-date">Tuesday, 7/16/19</span><div class="daySeparator-line"></div></div><div class="mediocre-item is-text" data-scroll-id="note-part987"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part987" data-index="74" style="margin-left: 35px;">PCR Digestion (Restriction DIgest)</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part988"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part988" data-index="75" style="margin-left: 70px;">1 mL DNA</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part989"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part989" data-index="76" style="margin-left: 70px;">5 mL NEB Buffer 2.1</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part990"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part990" data-index="77" style="margin-left: 70px;">1 ml EcoR1 HF</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part991"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part991" data-index="78" style="margin-left: 70px;">1 mL Pst 1</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part992"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part992" data-index="79" style="margin-left: 70px;">50 mL MGBW</div></div></div><div class="daySeparator" data-scroll-id="entryDay878"><div class="daySeparator-inputWrapper"><input class="daySeparator-inputWrapper-hiddenInput" type="input"/></div><span class="daySeparator-date">Thursday, 7/18/19</span><div class="daySeparator-line"></div></div><div class="mediocre-item is-text" data-scroll-id="note-part993"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part993" data-index="80" style="margin-left: 35px;">DIstribution Kit 2019</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part994"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part994" data-index="81" style="margin-left: 35px;">Transformation:</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part995"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part995" data-index="82" style="margin-left: 70px;">2 types of transformations: Electric Shock and Heat Shock</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part996"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part996" data-index="83" style="margin-left: 70px;">Wash cell in buffer solution to weaken the cell walls</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part997"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part997" data-index="84" style="margin-left: 35px;">Mini Prep: (Mini Prep kit + Monarch)</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part998"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part998" data-index="85" style="margin-left: 70px;">Spin colony to get the pellet on the bottom. Pellet 1-5 mL</div></div></div><div class="daySeparator" data-scroll-id="entryDay880"><div class="daySeparator-inputWrapper"><input class="daySeparator-inputWrapper-hiddenInput" type="input"/></div><span class="daySeparator-date">Monday, 7/22/19</span><div class="daySeparator-line"></div></div><div class="mediocre-item is-text" data-scroll-id="note-part999"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part999" data-index="86" style="margin-left: 35px;">Overnight Prep For Mini Prep (Grow Colonies)</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1000"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1000" data-index="87" style="margin-left: 70px;">5000 microliters of LB</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1001"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1001" data-index="88" style="margin-left: 70px;">5 microliters Chlor</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1002"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1002" data-index="89" style="margin-left: 70px;">10 microliters colony PCR</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1003"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 105px;"><span>■</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1003" data-index="90" style="margin-left: 105px;">1. Mix everything together</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1004"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 105px;"><span>■</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1004" data-index="91" style="margin-left: 105px;">2. Parafilm the tube</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1005"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 105px;"><span>■</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1005" data-index="92" style="margin-left: 105px;">3. Put in the Rotissirie</div></div></div><div class="daySeparator" data-scroll-id="entryDay882"><div class="daySeparator-inputWrapper"><input class="daySeparator-inputWrapper-hiddenInput" type="input"/></div><span class="daySeparator-date">Thursday, 7/25/19</span><div class="daySeparator-line"></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1006"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1006" data-index="93" style="margin-left: 35px;">Transformation for Red Colonies</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1007"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1007" data-index="94" style="margin-left: 35px;">Plasmid RFP + AMP </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1008"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1008" data-index="95" style="margin-left: 35px;"> DNA Distribution Kit</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1009"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1009" data-index="96" style="margin-left: 70px;">Linearized back bones, competent cell test kit, measurement kit</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1010"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1010" data-index="97" style="margin-left: 35px;">Reporter quantitizes how bright the colors are</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1011"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1011" data-index="98" style="margin-left: 35px;">Insert conatins ribosome, binding site</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1012"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1012" data-index="99" style="margin-left: 35px;">Bacterial Transformation: Method of foreign DNA being introduced into competent cells. Bacteria takes up the plasmids and expresses the specific gene in the DNA</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1013"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1013" data-index="100" style="margin-left: 35px;">Colony PCR: </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1014"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1014" data-index="101" style="margin-left: 70px;">Inside Thermocycler:</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1015"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 105px;"><span>■</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1015" data-index="102" style="margin-left: 105px;">Denaturation- separation of double stranded DNA</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1016"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 105px;"><span>■</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1016" data-index="103" style="margin-left: 105px;">Annealing- laying of primers on template DNA</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1017"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 105px;"><span>■</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1017" data-index="104" style="margin-left: 105px;">Extension- polymerase synthesizes new strands of DNA using dNTP's</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1018"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1018" data-index="105" style="margin-left: 70px;">Check product by running a .7% gel</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1019"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1019" data-index="106" style="margin-left: 70px;">Combine colonies with LB broth and antibiotics</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1020"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1020" data-index="107" style="margin-left: 70px;">Rotate overnight</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1021"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1021" data-index="108" style="margin-left: 70px;">Mini prep </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1022"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 105px;"><span>■</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1022" data-index="109" style="margin-left: 105px;">Method of extracting and purifying the plasmid DNA from bacterial cells. Results in large source of DNA that is pure and contains desired construction in a circulated form used for bacterial transformation or PCR reaction</div></div></div><div class="daySeparator" data-scroll-id="entryDay884"><div class="daySeparator-inputWrapper"><input class="daySeparator-inputWrapper-hiddenInput" type="input"/></div><span class="daySeparator-date">Tuesday, 7/30/19</span><div class="daySeparator-line"></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1023"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1023" data-index="110" style="margin-left: 35px;">Another transformation</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1024"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1024" data-index="111" style="margin-left: 35px;">Overnight prep (we want more bacteria with the plasmid)</div></div></div><div class="daySeparator" data-scroll-id="entryDay886"><div class="daySeparator-inputWrapper"><input class="daySeparator-inputWrapper-hiddenInput" type="input"/></div><span class="daySeparator-date">Wednesday, 7/31/19</span><div class="daySeparator-line"></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1025"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1025" data-index="112" style="margin-left: 35px;">Transcription of DNA in bacteria</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1026"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1026" data-index="113" style="margin-left: 70px;">DNA to RNA to Protien</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1027"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1027" data-index="114" style="margin-left: 35px;">Mini Prep (followed given instructions)</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1028"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1028" data-index="115" style="margin-left: 35px;">Nanodropped the concentrations made</div></div></div><div class="daySeparator" data-scroll-id="entryDay888"><div class="daySeparator-inputWrapper"><input class="daySeparator-inputWrapper-hiddenInput" type="input"/></div><span class="daySeparator-date">Thursday, 8/1/19</span><div class="daySeparator-line"></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1029"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1029" data-index="116" style="margin-left: 35px;">Transformation (GFP + AMP) and Gel Electrophorises</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1030"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1030" data-index="117" style="margin-left: 35px;">RNAi</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1031"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1031" data-index="118" style="margin-left: 70px;">Used to control genes</div></div></div><div class="daySeparator" data-scroll-id="entryDay890"><div class="daySeparator-inputWrapper"><input class="daySeparator-inputWrapper-hiddenInput" type="input"/></div><span class="daySeparator-date">Monday, 8/12/19</span><div class="daySeparator-line"></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1032"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1032" data-index="119" style="margin-left: 35px;">Transformed Ecoli cells with Gblock+Chlor plasmids</div></div></div><div class="daySeparator" data-scroll-id="entryDay892"><div class="daySeparator-inputWrapper"><input class="daySeparator-inputWrapper-hiddenInput" type="input"/></div><span class="daySeparator-date">Tuesday, 8/13/19</span><div class="daySeparator-line"></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1033"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1033" data-index="120" style="margin-left: 35px;">Looked at plates from transformation on 8/12</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1034"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1034" data-index="121" style="margin-left: 35px;">We had a few colonies that succesfully transformed</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1035"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1035" data-index="122" style="margin-left: 35px;">Took colonies from each plate and suspended them in seperate tubes with 20 microliters of MBGW (template)</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1036"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1036" data-index="123" style="margin-left: 70px;">Tubes were labeled S1, S2, &amp; S3, corresponding to plates that colonies were sourced from</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1037"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1037" data-index="124" style="margin-left: 35px;">Performed PCR with 1 micro templates from </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1038"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1038" data-index="125" style="margin-left: 70px;">12.5 microliters of Q5 Master Mix</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1039"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1039" data-index="126" style="margin-left: 70px;">1.5 microliters of VR Primer</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1040"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1040" data-index="127" style="margin-left: 70px;">1.5 microliters of VF2 Primer</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1041"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1041" data-index="128" style="margin-left: 70px;">1 microliter of the template</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1042"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1042" data-index="129" style="margin-left: 70px;">8.5 microliters of MBGW</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1043"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1043" data-index="130" style="margin-left: 35px;">Loaded test tubest with Colony PCR reagents into the Thermocycler</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1044"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1044" data-index="131" style="margin-left: 35px;">Cast a 0.7% Agarose gel to perform Gel electrophoresis with products from our PCR</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1045"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1045" data-index="132" style="margin-left: 35px;">Replenished 1x working stock of TAE from 50x stock</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1046"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1046" data-index="133" style="margin-left: 70px;">Performed C<sub>1</sub>V<sub>1</sub> = C<sub>2</sub>V<sub>2 </sub></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1047"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 105px;"><span>■</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1047" data-index="134" style="margin-left: 105px;">(50) V<sub>1</sub> = (1) 1000 mL</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1048"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 105px;"><span>■</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1048" data-index="135" style="margin-left: 105px;">V<sub>1</sub> = 20</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1049"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1049" data-index="136" style="margin-left: 70px;">Added 20 mL of 50x TAE stock into 980 mL of dH2O to create 1000 mL of 1x TAE working stock</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1050"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1050" data-index="137" style="margin-left: 35px;">Mixed PCR products with gel</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1051"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1051" data-index="138" style="margin-left: 35px;">Ran gel </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1052"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1052" data-index="139" style="margin-left: 35px;">No bands </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1053"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1053" data-index="140" style="margin-left: 35px;">Was most likely due to the fact that we did not spin down the the PCR reagents in their tubes prior to loading into thermocycler </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1054"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1054" data-index="141" style="margin-left: 35px;">Will Evaluate tonight  </div></div></div><div class="daySeparator" data-scroll-id="entryDay894"><div class="daySeparator-inputWrapper"><input class="daySeparator-inputWrapper-hiddenInput" type="input"/></div><span class="daySeparator-date">Wednesday, 8/14/19</span><div class="daySeparator-line"></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1055"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1055" data-index="142" style="margin-left: 35px;">Performed mini preparation of plasmid DNA: GFP + C, RFP + A </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1056"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1056" data-index="143" style="margin-left: 35px;">Repeatedly centrifuged and added wash buffers to DNA in order to isolate plasmid DNA from bacteria</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1057"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1057" data-index="144" style="margin-left: 70px;">Known as alkaline lysis </div></div></div><div class="daySeparator" data-scroll-id="entryDay896"><div class="daySeparator-inputWrapper"><input class="daySeparator-inputWrapper-hiddenInput" type="input"/></div><span class="daySeparator-date">Thursday, 8/15/19</span><div class="daySeparator-line"></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1058"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1058" data-index="145" style="margin-left: 35px;">Made and poured plates</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1059"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1059" data-index="146" style="margin-left: 70px;">Mixed 8.75 grams of agar with 250 milliters of distilled water</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1060"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1060" data-index="147" style="margin-left: 70px;">Put the mixture in a pressure cooker for 30 minutes</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1061"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1061" data-index="148" style="margin-left: 70px;">Let the mixture cool down for ten minutes</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1062"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1062" data-index="149" style="margin-left: 70px;">Add antibiotic to the mixture </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1063"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1063" data-index="150" style="margin-left: 70px;">Pour mixture onto plates</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1064"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1064" data-index="151" style="margin-left: 35px;">Performed a transformation using Ecoli T7 Express cells</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1065"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1065" data-index="152" style="margin-left: 35px;">Performed transformation on GFP + C made in mini prep on 8/14 to make sure we labled the tubes correctly </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1066"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1066" data-index="153" style="margin-left: 35px;">Constitutive promoter: always displaying trait, inducible promoters, repressible promoters</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1067"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1067" data-index="154" style="margin-left: 35px;">Initiation - Elongation - Termination (independent hairpin loop forms) </div></div></div><div class="daySeparator" data-scroll-id="entryDay898"><div class="daySeparator-inputWrapper"><input class="daySeparator-inputWrapper-hiddenInput" type="input"/></div><span class="daySeparator-date">Monday, 8/19/19</span><div class="daySeparator-line"></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1068"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1068" data-index="155" style="margin-left: 35px;">We found out that  while ligating out backbone folded onto itself. This prevented out insert from ligating with the backbone to form a full plasmid. To troubleshoot this we decided to use an enzyme called "Antartic Phosphatase" to cut the ends of the backbone. This will dephophate the backbone and prevent it from folding onto itself.</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1069"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1069" data-index="156" style="margin-left: 35px;">Dephosphating Backbone - 2microliter buffer, 1 microliter ant(Antartic Phosphatase), 10 microliter backbone, 7 mucroliter mgbw</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1070"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1070" data-index="157" style="margin-left: 35px;">Ligation- 2 microliter buffer, 1 microliter dephosphated DNA, 1 microliter ligase, 16 microliter insert</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1071"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 0px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1071" data-index="158" style="margin-left: 0px;"></div></div></div><div class="daySeparator" data-scroll-id="entryDay900"><div class="daySeparator-inputWrapper"><input class="daySeparator-inputWrapper-hiddenInput" type="input"/></div><span class="daySeparator-date">Tuesday, 8/20/19</span><div class="daySeparator-line"></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1072"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1072" data-index="159" style="margin-left: 35px;">We are transforming out newly made DNA from 8/19. If out dephophatase worked then the cells should grow with a blue flourescent color.</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1073"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1073" data-index="160" style="margin-left: 70px;">Take out E-Coli(c2987) cells and defrost them from nitrogen.</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1074"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1074" data-index="161" style="margin-left: 70px;">Put 4 chlor pretri dishes in an incubator and make an ice bath.</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1075"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1075" data-index="162" style="margin-left: 70px;">Pipette 50uL of E-Coli and put them into tubeG-Block 1, G-Block 2, G-Block 3, and  RFP control.</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1076"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1076" data-index="163" style="margin-left: 70px;">Pipette our designated plasmids onto each of our tube. 3uL for G-Block, 1uL for RFP.</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1077"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1077" data-index="164" style="margin-left: 70px;">Heat shock the tubes for 45secs at 42 ddegrees.</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1078"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1078" data-index="165" style="margin-left: 70px;">Take them out and put them in icebath for 10 min.</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1079"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1079" data-index="166" style="margin-left: 70px;">Add 950 uL of LB to the tubes.</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1080"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1080" data-index="167" style="margin-left: 70px;">Put parafilm on the tube and place in rotisserie for a hour.</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1081"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1081" data-index="168" style="margin-left: 35px;"></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1082"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 0px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1082" data-index="169" style="margin-left: 0px;"></div></div></div><div class="daySeparator" data-scroll-id="entryDay902"><div class="daySeparator-inputWrapper"><input class="daySeparator-inputWrapper-hiddenInput" type="input"/></div><span class="daySeparator-date">Tuesday, 8/27/19</span><div class="daySeparator-line"></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1083"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1083" data-index="170" style="margin-left: 35px;">Pasmid Prep (Mini Prep) for  blue chromo protein</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1084"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1084" data-index="171" style="margin-left: 35px;">Gel electrophoresis from the result of the plasmid prep</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1085"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 0px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1085" data-index="172" style="margin-left: 0px;"></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1086"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 0px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1086" data-index="173" style="margin-left: 0px;"></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1087"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 0px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1087" data-index="174" style="margin-left: 0px;"></div></div></div><div class="daySeparator" data-scroll-id="entryDay904"><div class="daySeparator-inputWrapper"><input class="daySeparator-inputWrapper-hiddenInput" type="input"/></div><span class="daySeparator-date">Tuesday, 9/3/19</span><div class="daySeparator-line"></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1088"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 0px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1088" data-index="175" style="margin-left: 0px;">Workflow for the day was to inoculate colonies from separate plasmid cassette transformation into overnight culture, that will be used to create individual plasmid preps for Wednesday 9/4.</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1089"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1089" data-index="176" style="margin-left: 35px;"><span style="color:#222222"><span style="background-color:#FFFFFF">First we counted all the colonies on the petre dishes, and got tubes for each colony (24). </span></span></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1090"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1090" data-index="177" style="margin-left: 35px;"><span style="color:#222222"><span style="background-color:#FFFFFF">Next we placed the 3 millimeters of Lb +Chlor. </span></span></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1091"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1091" data-index="178" style="margin-left: 35px;"><span style="color:#222222"><span style="background-color:#FFFFFF">Then we scraped each of the colonies off of the petre dishes using the loops and placed them into the tubes of Lb + Chlor. </span></span></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1092"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1092" data-index="179" style="margin-left: 35px;"><span style="color:#222222"><span style="background-color:#FFFFFF">Then we used film to seal Oof the tubes and placed them into the shaker incubator to grow up overnight.</span></span></div></div></div><div class="daySeparator" data-scroll-id="entryDay906"><div class="daySeparator-inputWrapper"><input class="daySeparator-inputWrapper-hiddenInput" type="input"/></div><span class="daySeparator-date">Wednesday, 9/4/19</span><div class="daySeparator-line"></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1093"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 0px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1093" data-index="180" style="margin-left: 0px;">Plasmid Prep</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1094"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1094" data-index="181" style="margin-left: 35px;">take out 24 large tubes of Lb + Chlor AK and SOB </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1095"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1095" data-index="182" style="margin-left: 35px;">Pipette 2 microliter for each of the Lb + Chlor AK and SOB into the microcentrifuge tubes </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1096"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1096" data-index="183" style="margin-left: 35px;">place them balanced and spaced eqally in centrifugation for 30 seconds </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1097"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1097" data-index="184" style="margin-left: 35px;">Discard by pipetting liquid out and leave the cells at the bottom</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1098"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1098" data-index="185" style="margin-left: 35px;">pipette 2 microliter of Lb + Chlor back inside the tube. Then repeat the process of placing in the centrifugation for 30 seconds</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1099"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1099" data-index="186" style="margin-left: 35px;">Pipette 200 microliter of Plasmid Resuspension Buffer into the tube and slowly break or mix with the cells. (until the solid is gone)</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1100"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1100" data-index="187" style="margin-left: 35px;">Add 200 microliter of Plasmid Lysis Buffer in the tube and shake it for 5 times. Set for 1 minute at a room temperature.</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1101"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1101" data-index="188" style="margin-left: 35px;">Add 400 microliter of Plasmid Neutralization Buffer in the tube and shake it until the color completely changes. Set for 2 minutes at room temperatur</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1102"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1102" data-index="189" style="margin-left: 35px;">Back to centrifugation (balanced and spaced equally) for 5 minutes.</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1103"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1103" data-index="190" style="margin-left: 35px;">Transfer the supernatant to the plasmid column and place it into the centrifugation for 4 minutes. </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1104"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1104" data-index="191" style="margin-left: 35px;">Transfer the column into a new tube and add 200 microliter of Plamid Wash Buffer 1. Then place it into the centrifugation for 1 minute.</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1105"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1105" data-index="192" style="margin-left: 35px;">Add 400 microliter of Plasmid Wash Buffer 2 into the column and place it into the centriguation for 1 minute. </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1106"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1106" data-index="193" style="margin-left: 35px;">Transfer the columns to a new microfuge tube and add 50 microliters of DNA Elution Buffer in the tube. Wait for 1 minute and place it into the centrifugation for 1 minute.</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1107"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1107" data-index="194" style="margin-left: 35px;">Separate the columns. The leftover liquid is the DNA sample.</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1108"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 0px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1108" data-index="195" style="margin-left: 0px;"></div></div></div><div class="daySeparator" data-scroll-id="entryDay908"><div class="daySeparator-inputWrapper"><input class="daySeparator-inputWrapper-hiddenInput" type="input"/></div><span class="daySeparator-date">Thursday, 9/5/19</span><div class="daySeparator-line"></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1109"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 0px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1109" data-index="196" style="margin-left: 0px;">Plasmid Prep through Gel </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1110"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1110" data-index="197" style="margin-left: 35px;">Put the weigh paper on a gram scale. Then measure 0.35 g of agarose. </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1111"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1111" data-index="198" style="margin-left: 35px;">50mL of 1X TAE and put it into a erlenmeyer flask. Put the 0.35 g of agarose in the flask and slowly swirl / mix it. </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1112"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1112" data-index="199" style="margin-left: 35px;">cover the top of the elenmeyer flask with a clean wrap foil and poke the top. </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1113"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1113" data-index="200" style="margin-left: 35px;">Put it into a microwave for 30 seconds.</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1114"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1114" data-index="201" style="margin-left: 35px;">Then slowly swirl it and put it in the microwave again for 30 seconds.</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1115"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1115" data-index="202" style="margin-left: 35px;">Then slowly swril it and put it in the microwave for 15 seconds. Cool it after it's done. </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1116"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1116" data-index="203" style="margin-left: 35px;">Prepare the Gel electrophoresis / Gel box. Then pour the agarose liquid into the Gel box and cool it for a while.</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1117"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1117" data-index="204" style="margin-left: 35px;">After cooling, put 5 microliter of Bromide in the gel liquid and swirl / mix it. </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1118"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1118" data-index="205" style="margin-left: 35px;">Place the comb and make the wells. Wait for 30 minutes. </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1119"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1119" data-index="206" style="margin-left: 35px;">Replace the Gel and put a black piece of paper under the gel box for better observations. </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1120"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1120" data-index="207" style="margin-left: 35px;">pour the 1X TAE and fill the gel box until it covers the gel.</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1121"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1121" data-index="208" style="margin-left: 35px;">prepare the parafilms, ladder, purple dye, and the AK and SOB DNA samples. </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1122"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1122" data-index="209" style="margin-left: 35px;">Then pipette 6 microliter of ladder and slowly + gently place it in one of the wells. </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1123"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1123" data-index="210" style="margin-left: 35px;">Then pipette 1 microliter of purple dye and place it on the parafilm. </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1124"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1124" data-index="211" style="margin-left: 35px;">add 5 microliter of one of the DNA samples and mix it with the purple dye.</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1125"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1125" data-index="212" style="margin-left: 35px;">Then pipette as 6 microliter of the mixed purple dye and DNA sample and place it to the next well of the ladder. </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1126"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1126" data-index="213" style="margin-left: 35px;">*repeat the process for each of the DNA samples. </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1127"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1127" data-index="214" style="margin-left: 35px;">After filling the wells, carry it to the power source and set as 80V. Put the cover on top of the gel box and set it as 'run' for 65minutes. </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1128"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1128" data-index="215" style="margin-left: 35px;">After 65 minutes, take the gel out and pour out the liquid.</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1129"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1129" data-index="216" style="margin-left: 35px;">Put the gel in the UV transilluminator and turn the power on. </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1130"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 0px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1130" data-index="217" style="margin-left: 0px;"></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1131"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 0px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1131" data-index="218" style="margin-left: 0px;"></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1132"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 0px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1132" data-index="219" style="margin-left: 0px;"></div></div></div></div></div><div class="dnd-drop-container-border"></div></div></div><div class="noteEditor-nueModuleContainer"></div></div></div><div class="printable-page-container"><div class="protocolEditor"><div></div><div class="protocolEditor-pageContainer"><div class="protocolEditor-page"><div class="protocolEditor-contentWrapper"><h1 class="mediocre-titleEditor is-locked"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="false" data-cid="ep1161-title" data-index="0">Sequential Double Digest</div></h1><div><h2>Introduction</h2><div class="intro-editor" data-editor="intro"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="true" data-cid="ep1161-intro" data-index="0">This is the Sequential Double Digest Protocol with Standard Restriction Enzymes. If there is no buffer in which the two enzymes exhibit &gt; 50% activity, this sequential digest can be performed.
+More information from NEB can be found <a href="https://www.neb.com/tools-and-resources/usage-guidelines/double-digests" rel="nofollow noopener noreferrer" target="_blank">here.</a>
-<div class="column full_size">
+Double Digests can be designed using <a href="https://www.neb.com/tools-and-resources/interactive-tools/double-digest-finder" rel="nofollow noopener noreferrer" target="_blank">NEB's Double Digest Finder</a>.
-<h1>Notebook</h1>
+<a href="http://nebcloner.neb.com/#!/" rel="nofollow noopener noreferrer" target="_blank">NEBcloner</a> will help guide your reaction buffer selection when setting up double digests.
-<p> Below is the documentation of our daily lab work as well as our protocols.</p>
-<p> 9/27/2019</p>
-<p>BwoB iGEM 2019</p>
+See the <a href="https://www.neb.com/tools-and-resources/usage-guidelines/nebuffer-performance-chart-with-restriction-enzymes" rel="nofollow noopener noreferrer" target="_blank">NEBuffer Activity/Performance Chart with Restriction Enzymes</a> for the incubation temperatures.
-<p>Project: 2019 iGEM</p>
-<p>Authors: Ellen Jorgensen</p>
-<p>FRIDAY, 5/24/19</p>
-<p>Mentor: Craig</p>
-<p>PI: Ellen</p>
-<p>Administrator: Chris</p>
-<p>&#9679;Introductions of members</p>
-<p>&#9679;Biobuilder: download Riley</p>
-<p>&#9679;iGEM Academy on Youtube</p>
-<p>&#9675;Collection of videos: parodies of molecular biology</p>
-<p>&#9675;Helpful for a basis</p>
-<p>&#9679;Introduce iGEM competition description to BEAM center students</p>
-<p>&#9679;Restriction Digest</p>
-<p>&#9675;2017: targeted psyllid (vector) so it&rsquo;d have trouble carrying virus</p>
-<p>&#9675;Talking Biotech Podcast 185 by Dr. Steven Savage</p>
-<p>&#9632;Reach out about citrus virus -- use Tristeza virus</p>
-<p>&#9675;Tristeza: 1960s virus mutated inside citrus plant</p>
-<p>&#9679;DNA Learning Center: educational videos of crystallography</p>
-<p>&#9679;Intro to pipette usage -- hands-on practice</p>
-<p>FRIDAY, 5/31/19</p>
-<p>&#9679;Watched video on recombinant DNA (dnalc-mechanisms of recombination)</p>
-<p>&#9679;Cut plasmids with restriction enzymes</p>
-<p>FRIDAY, 6/7/19</p>
-<p>&#9679;Showed video to introduce restriction enzyme</p>
-<p>&#9679;Displayed process for agarose gel for electrophoresis (1% gel = 0.5 TAE Solution)</p>
-<p>&#9679;Demonstrated how to prep gel, pour it, and add ethidium bromide</p>
-<p>&#9675;Restriction digest products still in the fridge</p>
-<p>FRIDAY, 6/14/19</p>
-<p>&#9679;Introduced Google Classroom as main communication platform</p>
-<p>&#9675;Google Hangout &amp; Discord when unable to attend in person -- useful video broadcast over channels</p>
-<p>&#9675;Four interns in lab to mentor students</p>
-<p>&#9679;Safety lab forms: for iGEM and Biotech Without Borders</p>
-<p>&#9679;Review Restriction Digest: enzymes = Bozeman Science video</p>
-<p>&#9679;Polymerase Chain Reaction (PCR)</p>
-<p>&#9679;Walk through and introduced basic lab</p>
-<p>FRIDAY, 6/21/19</p>
-<p>&#9679;Safety agreements &amp; forms (1): If under 18, need signatures of parents</p>
-<p>&#9675;Classroom link with online version available</p>
-<p>&#9679;Deliverables on iGEM ~ test: answer questions</p>
-<p>&#9675;Team wiki (see 2017 fancy team wiki), team poster, team presentation, judging form, project attributors, safety, registry part pages, project inspiration</p>
-<p>&#9679;iGEM account: igem.org &rarr; fill out personal info as prompted &rarr; login to account &rarr; membership code to team: 327718</p>
-<p>&#9675;Pending queue -- need verification</p>
-<p>&#9679;Keep up with Google Classroom account (3); Biotech Without Borders (4) forms</p>
-<p>&#9679;Video call with Dr. Georgios Vidalakis of the University of California, Riverside</p>
-<p>&#64257;le:///tmp/tmppp6q64.html</p>
-<p>1/12</p>
-<p> 9/27/2019</p>
-<p>BwoB iGEM 2019 &middot; Benchling</p>
-<p>&#9675;1998-2005: FL neglected severity of HLB insect - disease spread and threatens the citrus industry; FL has lost half the</p>
-<p>industry; 2008: CA industries jeopardized</p>
-<p>&#9675;Improve RATE of detection of bacteria in leaves; create a quiz that allows kids across the country to help diagnose millions of leaves (virtual game); trap the psyllids with traps (acetic acid); infrared cameras track disease</p>
-<p>&#9675;Localized immunity issues? signals are systemic; surrogate bacteria in mango (cultures that can be sent over) ALSO can send over actual bacteria Diaphorina citri after arranging the permits; california suspecting peptides &amp; concern with consumers; smaller virus RNA -> easier to work with the tristeza virus</p>
-<p>&#9675;https://news.ucr.edu/articles/2019/04/01/plant-pathologist-leads-research-stop-spread-citrus-destroying-disease</p>
-<p>&#9679;See Craig for head start with lab work &rarr; increases expertise &amp; familiarity</p>
-<p>&#9675;Start with scheduling with students -- genetic engineering tasks</p>
-<p>FRIDAY, 6/28/19</p>
-<p>&#9679;Established that the actual bacteria Candidatus Liberibacter asiaticus would not be easily culturable</p>
-<p>&#9679;Considered periwinkle as a cultured plant &amp; discussed the surrogate bacteria as usable: Liberibacter crescens</p>
-<p>&#9679;Reviewed restriction enzyme &amp; bioengineering techniques with our team members</p>
-<p>&#9679;Established that we will create a written protocol for new techniques for members away at Guatemala</p>
-<p>&#9679;Introduced t-shirt design project &amp; team project names</p>
-<p>MONDAY, 7/8/19</p>
-<p>&#9679;Practice lab techniques</p>
-<p>&#9675;Pipet</p>
-<p>&#9675;Ligation</p>
-<p>&#9675;Colony PCR</p>
-<p>&#9675;Gel Electrophoresis</p>
-<p>&#9675;Transformation</p>
-<p>&#9675;Restriction Digest</p>
-<p>TUESDAY, 7/9/19</p>
-<p>&#9679;PCR Polymerase Chain Reaction</p>
-<p>&#9675;Amplify a certain area of DNA</p>
-<p>&#9632;1. Break Apart Double Helix (Heat it up)- Denaturing</p>
-<p>&#9632;2. Primers sit on complementary base pair</p>
-<p>&#9632;3. Extend the areas after primers</p>
-<p>&#9679;1 X TAE contents:</p>
-<p>&#9675; C1V1=C2V2 (C is concentration, V is volume)</p>
-<p>&#9679;Gel Electrophoresis:</p>
-<p>&#9675;1. Pour out 50 mL 1 X TAE into flask</p>
-<p>&#9675;2. Add .35 g agarose</p>
-<p>&#9675;3. Cover Flask with Plastic Wrap</p>
-<p>&#9675;4. Put in microwave for 1 minute 15 seconds (check every 30 seconds)</p>
-<p>&#9675;5. Pour into gel mold and add 5 microliters of ethidium bromide</p>
-<p>&#9675;6. Put in combs and let sit for 30 minutes</p>
-<p>FRIDAY, 7/12/19</p>
-<p>&#9679;Remove syllids as they pick up RNAi + cloning project</p>
-<p>&#9679;Fluorescent protein genes picked out</p>
-<p>&#9679;iGEM BioBrick</p>
-<p>&#57666;iGEM_Meeting_Notebook.pdf</p>
-<p>TUESDAY, 7/16/19</p>
-<p>&#9679; PCR Digestion (Restriction DIgest)</p>
-<p>&#64257;le:///tmp/tmppp6q64.html</p>
-<p>2/12</p>
-<p> 9/27/2019</p>
-<p>BwoB iGEM 2019 &middot; Benchling</p>
-<p>&#9675;1 mL DNA</p>
-<p>&#9675;5 mL NEB Buffer 2.1</p>
-<p>&#9675;1 ml EcoR1 HF</p>
-<p>&#9675;1 mL Pst 1</p>
-<p>&#9675;50 mL MGBW</p>
-<p>THURSDAY, 7/18/19</p>
-<p>&#9679;DIstribution Kit 2019</p>
-<p>&#9679;Transformation:</p>
-<p>&#9675;2 types of transformations: Electric Shock and Heat Shock</p>
-<p>&#9675;Wash cell in buffer solution to weaken the cell walls</p>
-<p>&#9679;Mini Prep: (Mini Prep kit + Monarch)</p>
-<p>&#9675;Spin colony to get the pellet on the bottom. Pellet 1-5 mL</p>
-<p>MONDAY, 7/22/19</p>
-<p>&#9679;Overnight Prep For Mini Prep (Grow Colonies)</p>
-<p>&#9675;5000 microliters of LB</p>
-<p>&#9675;5 microliters Chlor</p>
-<p>&#9675;10 microliters colony PCR</p>
-<p>&#9632;1. Mix everything together</p>
-<p>&#9632;2. Parafilm the tube</p>
-<p>&#9632;3. Put in the Rotissirie</p>
-<p>THURSDAY, 7/25/19</p>
-<p>&#9679;Transformation for Red Colonies</p>
-<p>&#9679;Plasmid RFP + AMP</p>
-<p>&#9679;DNA Distribution Kit</p>
-<p>&#9675;Linearized back bones, competent cell test kit, measurement kit</p>
-<p>&#9679;Reporter quantitizes how bright the colors are</p>
-<p>&#9679;Insert conatins ribosome, binding site</p>
-<p>&#9679;Bacterial Transformation: Method of foreign DNA being introduced into competent cells. Bacteria takes up the plasmids and expresses the specific gene in the DNA</p>
-<p>&#9679;Colony PCR:</p>
-<p>&#9675;Inside Thermocycler:</p>
-<p>&#9632;Denaturation- separation of double stranded DNA</p>
-<p>&#9632;Annealing- laying of primers on template DNA</p>
-<p>&#9632;Extension- polymerase synthesizes new strands of DNA using dNTP's</p>
-<p>&#9675;Check product by running a .7% gel</p>
-<p>&#9675;Combine colonies with LB broth and antibiotics</p>
-<p>&#9675;Rotate overnight</p>
-<p>&#9675;Mini prep</p>
-<p>&#9632;Method of extracting and purifying the plasmid DNA from bacterial cells. Results in large source of DNA that is pure and contains desired construction in a circulated form used for bacterial transformation or PCR reaction</p>
-<p>TUESDAY, 7/30/19</p>
-<p>&#9679;Another transformation</p>
-<p>&#9679;Overnight prep (we want more bacteria with the plasmid)</p>
-<p>WEDNESDAY, 7/31/19</p>
-<p>&#9679;Transcription of DNA in bacteria</p>
-<p>&#9675;DNA to RNA to Protien</p>
-<p>&#9679;Mini Prep (followed given instructions)</p>
-<p>&#9679;Nanodropped the concentrations made</p>
-<p>&#64257;le:///tmp/tmppp6q64.html</p>
-<p>3/12</p>
-<p> 9/27/2019</p>
-<p>BwoB iGEM 2019 &middot; Benchling</p>
-<p>THURSDAY, 8/1/19</p>
-<p>&#9679;Transformation (GFP + AMP) and Gel Electrophorises</p>
-<p>&#9679;RNAi</p>
-<p>&#9675;Used to control genes</p>
-<p>MONDAY, 8/12/19</p>
-<p>&#9679; Transformed Ecoli cells with Gblock+Chlor plasmids</p>
-<p>TUESDAY, 8/13/19</p>
-<p>&#9679;Looked at plates from transformation on 8/12</p>
-<p>&#9679;We had a few colonies that succesfully transformed</p>
-<p>&#9679;Took colonies from each plate and suspended them in seperate tubes with 20 microliters of MBGW (template)</p>
-<p>&#9675;Tubes were labeled S1, S2, &amp; S3, corresponding to plates that colonies were sourced from</p>
-<p>&#9679;Performed PCR with 1 micro templates from</p>
-<p>&#9675;12.5 microliters of Q5 Master Mix</p>
-<p>&#9675;1.5 microliters of VR Primer</p>
-<p>&#9675;1.5 microliters of VF2 Primer</p>
-<p>&#9675;1 microliter of the template</p>
-<p>&#9675;8.5 microliters of MBGW</p>
-<p>&#9679;Loaded test tubest with Colony PCR reagents into the Thermocycler</p>
-<p>&#9679;Cast a 0.7% Agarose gel to perform Gel electrophoresis with products from our PCR</p>
-<p>&#9679;Replenished 1x working stock of TAE from 50x stock</p>
-<p>&#9675;Performed C1V1 = C2V2</p>
-<p>&#9632;(50) V1 = (1) 1000 mL</p>
-<p>&#9632;V1 = 20</p>
-<p>&#9675;Added 20 mL of 50x TAE stock into 980 mL of dH2O to create 1000 mL of 1x TAE working stock</p>
-<p>&#9679;Mixed PCR products with gel</p>
-<p>&#9679;Ran gel</p>
-<p>&#9679;No bands</p>
-<p>&#9679;Was most likely due to the fact that we did not spin down the the PCR reagents in their tubes prior to loading into thermocycler</p>
-<p>&#9679;Will Evaluate tonight</p>
-<p>WEDNESDAY, 8/14/19</p>
-<p>&#9679;Performed mini preparation of plasmid DNA: GFP + C, RFP + A</p>
-<p>&#9679;Repeatedly centrifuged and added wash buffers to DNA in order to isolate plasmid DNA from bacteria</p>
-<p>&#9675;Known as alkaline lysis</p>
-<p>THURSDAY, 8/15/19</p>
-<p>&#9679;Made and poured plates</p>
-<p>&#9675;Mixed 8.75 grams of agar with 250 milliters of distilled water</p>
-<p>&#9675;Put the mixture in a pressure cooker for 30 minutes</p>
-<p>&#9675;Let the mixture cool down for ten minutes</p>
-<p>&#9675;Add antibiotic to the mixture</p>
-<p>&#9675;Pour mixture onto plates</p>
-<p>&#9679;Performed a transformation using Ecoli T7 Express cells</p>
-<p>&#9679;Performed transformation on GFP + C made in mini prep on 8/14 to make sure we labled the tubes correctly</p>
-<p>&#9679;Constitutive promoter: always displaying trait, inducible promoters, repressible promoters</p>
-<p>&#9679;Initiation - Elongation - Termination (independent hairpin loop forms)</p>
-<p>MONDAY, 8/19/19</p>
-<p>&#9679;We found out that while ligating out backbone folded onto itself. This prevented out insert from ligating with the backbone to form a full plasmid. To troubleshoot this we decided to use an enzyme called &quot;Antartic Phosphatase&quot; to cut the ends of the backbone. This will dephophate the backbone and prevent it from folding onto itself.</p>
-<p>&#64257;le:///tmp/tmppp6q64.html</p>
-<p>4/12</p>
-<p> 9/27/2019</p>
-<p>BwoB iGEM 2019 &middot; Benchling</p>
-<p>&#9679;Dephosphating Backbone - 2microliter buffer, 1 microliter ant(Antartic Phosphatase), 10 microliter backbone, 7 mucroliter mgbw</p>
-<p>&#9679;Ligation- 2 microliter buffer, 1 microliter dephosphated DNA, 1 microliter ligase, 16 microliter insert</p>
-<p>TUESDAY, 8/20/19</p>
-<p>&#9679;We are transforming out newly made DNA from 8/19. If out dephophatase worked then the cells should grow with a blue flourescent color.</p>
-<p>&#9675;Take out E-Coli(c2987) cells and defrost them from nitrogen.</p>
-<p>&#9675;Put 4 chlor pretri dishes in an incubator and make an ice bath.</p>
-<p>&#9675;Pipette 50uL of E-Coli and put them into tubeG-Block 1, G-Block 2, G-Block 3, and RFP control.</p>
-<p>&#9675;Pipette our designated plasmids onto each of our tube. 3uL for G-Block, 1uL for RFP.</p>
-<p>&#9675;Heat shock the tubes for 45secs at 42 ddegrees.</p>
-<p>&#9675;Take them out and put them in icebath for 10 min.</p>
-<p>&#9675;Add 950 uL of LB to the tubes.</p>
-<p>&#9675;Put parafilm on the tube and place in rotisserie for a hour.</p>
-<p>TUESDAY, 8/27/19</p>
-<p>&#9679;Pasmid Prep (Mini Prep) for blue chromo protein</p>
-<p>&#9679;Gel electrophoresis from the result of the plasmid prep</p>
-<p>TUESDAY, 9/3/19</p>
-<p>Workflow for the day was to inoculate colonies from separate plasmid cassette transformation into overnight culture, that will be used to create individual plasmid preps for Wednesday 9/4.</p>
-<p>&#9679;First we counted all the colonies on the petre dishes, and got tubes for each colony (24).</p>
-<p>&#9679;Next we placed the 3 millimeters of Lb +Chlor.</p>
-<p>&#9679;Then we scraped each of the colonies off of the petre dishes using the loops and placed them into the tubes of Lb + Chlor.</p>
-<p>&#9679;Then we used film to seal Oof the tubes and placed them into the shaker incubator to grow up overnight.</p>
-<p>WEDNESDAY, 9/4/19</p>
-<p>Plasmid Prep</p>
-<p>&#9679;take out 24 large tubes of Lb + Chlor AK and SOB</p>
-<p>&#9679;Pipette 2 microliter for each of the Lb + Chlor AK and SOB into the microcentrifuge tubes</p>
-<p>&#9679;place them balanced and spaced eqally in centrifugation for 30 seconds</p>
-<p>&#9679;Discard by pipetting liquid out and leave the cells at the bottom</p>
-<p>&#9679;pipette 2 microliter of Lb + Chlor back inside the tube. Then repeat the process of placing in the centrifugation for 30 seconds</p>
-<p>&#9679;Pipette 200 microliter of Plasmid Resuspension Buffer into the tube and slowly break or mix with the cells. (until the solid is gone)</p>
-<p>&#9679;Add 200 microliter of Plasmid Lysis Buffer in the tube and shake it for 5 times. Set for 1 minute at a room temperature.</p>
-<p>&#9679;Add 400 microliter of Plasmid Neutralization Buffer in the tube and shake it until the color completely changes. Set for 2 minutes at room temperatur</p>
-<p>&#9679;Back to centrifugation (balanced and spaced equally) for 5 minutes.</p>
-<p>&#9679;Transfer the supernatant to the plasmid column and place it into the centrifugation for 4 minutes.</p>
-<p>&#9679;Transfer the column into a new tube and add 200 microliter of Plamid Wash Buffer 1. Then place it into the centrifugation for 1 minute.</p>
-<p>&#9679;Add 400 microliter of Plasmid Wash Buffer 2 into the column and place it into the centriguation for 1 minute.</p>
-<p>&#9679;Transfer the columns to a new microfuge tube and add 50 microliters of DNA Elution Buffer in the tube. Wait for 1 minute and place it into the centrifugation for 1 minute.</p>
-<p>&#9679;Separate the columns. The leftover liquid is the DNA sample.</p>
-<p>&#64257;le:///tmp/tmppp6q64.html</p>
-<p>5/12</p>
-<p> 9/27/2019</p>
-<p>BwoB iGEM 2019 &middot; Benchling</p>
-<p>THURSDAY, 9/5/19</p>
-<p>Plasmid Prep through Gel</p>
-<p>&#9679;Put the weigh paper on a gram scale. Then measure 0.35 g of agarose.</p>
-<p>&#9679;50mL of 1X TAE and put it into a erlenmeyer flask. Put the 0.35 g of agarose in the flask and slowly swirl / mix it.</p>
-<p>&#9679;cover the top of the elenmeyer flask with a clean wrap foil and poke the top.</p>
-<p>&#9679;Put it into a microwave for 30 seconds.</p>
-<p>&#9679;Then slowly swirl it and put it in the microwave again for 30 seconds.</p>
-<p>&#9679;Then slowly swril it and put it in the microwave for 15 seconds. Cool it after it's done.</p>
-<p>&#9679;Prepare the Gel electrophoresis / Gel box. Then pour the agarose liquid into the Gel box and cool it for a while.</p>
-<p>&#9679;After cooling, put 5 microliter of Bromide in the gel liquid and swirl / mix it.</p>
-<p>&#9679;Place the comb and make the wells. Wait for 30 minutes.</p>
-<p>&#9679;Replace the Gel and put a black piece of paper under the gel box for better observations.</p>
-<p>&#9679;pour the 1X TAE and fill the gel box until it covers the gel.</p>
-<p>&#9679;prepare the parafilms, ladder, purple dye, and the AK and SOB DNA samples.</p>
-<p>&#9679;Then pipette 6 microliter of ladder and slowly + gently place it in one of the wells.</p>
-<p>&#9679;Then pipette 1 microliter of purple dye and place it on the parafilm.</p>
-<p>&#9679;add 5 microliter of one of the DNA samples and mix it with the purple dye.</p>
-<p>&#9679;Then pipette as 6 microliter of the mixed purple dye and DNA sample and place it to the next well of the ladder.</p>
-<p>&#9679;*repeat the process for each of the DNA samples.</p>
-<p>&#9679;After filling the wells, carry it to the power source and set as 80V. Put the cover on top of the gel box and set it as 'run' for 65minutes.</p>
-<p>&#9679;After 65 minutes, take the gel out and pour out the liquid.</p>
-<p>&#9679;Put the gel in the UV transilluminator and turn the power on.</p>
-<p>&#64257;le:///tmp/tmppp6q64.html</p>
-<p>6/12</p>
-</div>
-<div class="clear"></div>
-<div class="column two_thirds_size">
-<h3>What should this page have?</h3>
-<ul>
-<li>Chronological notes of what your team is doing.</li>
-<li> Brief descriptions of daily important events.</li>
-<li>Pictures of your progress. </li>
-<li>Mention who participated in what task.</li>
-</ul>
-<h2>Using DNA distribution kit plates: Rehydration of registry DNA from kit plate</h2>
-<p>Wells in the DNA distribution kit plates contain 2-3 ng of dry DNA. This should be enough DNA for a few transformation, but insufficient for immediate restriction and ligation.</p>
-<h4>Materials</h4>
-<ul style="list-style-type:disc;">
-<li>DNA distribution kit plates</li>
-<li>Sterile distilled water</li>
-<li>Micropipette</li>
-<li>Sterile micropipette tip</li>
-</ul>
-<h4>Protocol</h4>
-<ol>
-<li>Locate the well with the DNA part you need</li>
-<li>Using a micro pipette, draw 10 μL sterile distilled water into a sterile pipette tip</li>
-<li>Use the pipette tip to puncture a hole through the cover foil and into the well containing DNA you need.</li>
-<li>Release the water into the well and pipette up and down a few times to rehydrate the DNA you need</li>
-<li>Store the re-hydrated DNA in the -20C freezer. You can either leave it in the original plate, or transfer it into a separate tube (make sure to label the tube!).</li>
-<li>The rehydrated DNA can be used immediately to transform competent cells. As little as 1 μL should be enough for one transformation.</li>
-</ol>
-<h2>Restriction Digest</h2>
-<p>This protocol is for cutting approximately 500ng of plasmid with two different restriction enzymes. This should provide enough cut DNA for both ligation and agarose gel electrophoresis.</p>
-<h4>Materials</h4>
-<ul style="list-style-type:disc;">
-<li>Plasmid DNA</li>
-<li>10x Restriction buffet</li>
-<li>Sterile distilled water</li>
-<li>Restriction Enzymes(EcoR1, Spe1, Xba1, and Pst1)
-<li>Thermocycler</li>
-<li>(0.6 mL) Microcentrufuge tube</li>
-<li>Microcentrifuge</li>
-<li>Ice</li>
-<li>Micropipettes and Sterile pipette tips</li>
-<li>Water bath and 37C</li>
-</ul>
-<h4>Protocol</h4>
-<ol>
-<li>Thaw	all	solutions	and	buffers	needed.	Homogenize	the	contents	by	gently	flicking	the	tube.	If
-possible,	collect	all	liquid	at	bottom	of	tube	by	spinning	briefly	in	the	microcentrifuge	at	maximum
-speed.	Place	tubes	on	ice.</li>
-<li>Label	a	sterile	0.6	mL	microcentrifuge	tube	with	an	identifying	label	for	the	particular	restriction
-digestion	you’re	doing.	Make	sure	that	you	use	a	permanent	marker	and	the	tube	fits	inside	the
-heating	block	of	your	thermocycler.</li>
-<li>Pipette	the	following	into	the	tube	in	the	order	listed.	It	adds	up	to	a	total	volume	of	20	μL</li>
-<ol>
-<li>7	μL	sterile	distilled	water</li>
-<li>10	μL	plasmid	DNA	</li>
-<li>2	μL	10x	restriction	buffer	</li>
-<li>0.5	μL	Restriction	Enzyme	#1</li>
-<li>0.5	μL	Restriction	Enzyme	#2	</li>
-</ol>
-<li>Mix	gently	by	pipetting	up	and	down	a	few	times.	You	can	bring	all	the	liquid	to	the	bottom	of	the
-tube	by	spinning	it	briefly	in	the	microcentrifuge	at	maximum	speed.</li>
-<li>Incubate	at	37	°C	in	water	bath	or	thermocycler	for	30	minutes	to	1	hour.</li>
-<li>Incubate at	80	°C	for	20	minutes.	This	will	denature	the	restriction	enzymes,	which	otherwise	may
-interfere	with	subsequent	steps.</li>
-<li>Store	the	restriction	digests	in	- 20	°C	freezer,	or	proceed	immediately	with	ligation	and/or	agarose
-gel	electrophoresis.</li>
-</ol>
-<h2>Growing	bacteria	from	agar	stabs	onto	plates</h2>
-<p>This method allow us to grow bacteria colonies on pretri dishes</p>
-<h4>Materials</h4>
-<ul style="list-style-type:disc;">
-<li>agar	stabs	with	the	bacteria	you	wish	to	grow</li>
-<li>LB	agar	plates</li>
-<li>inoculating	loop</li>
-<li>Bunsen	burner</li>
-<li>incubator	37	°C</li>
-</ul>
-<h4>Protocol</h4>
-<ol>
-<li>Obtain	LB	agar	plates	suitable	for	the	bacteria	you	wish	to	grow.	If	bacteria	carry	plasmid	DNA	that
-makes	them	resistant	to	a	certain	antibiotic,	use	LB	agar	plates	containing	the	respective	antibiotic.
-For	E.	coli	with	no	antibiotic	resistance	(e.g.	NEB	10	beta	or	DH5	alpha	cells),	which	you	would	need
-for	making	competent	cells,	use	LB	agar	plates	without	any	antibiotics.</li>
-<li>Warm	up	the	plates	to	room	temperature,	and	label	them.</li>
-<li>Locate	the	sample	of	live	bacteria	inside	the	agar	stab.	Use	an	inoculating	loop	to	scoop	some	of	the
-bacteria	and	streak	them	onto	a	plate	in	a	zig-zag	line.	Be	gentle	when	drawing	the	line	to	avoid
-breaking	the	agar.</li>
-<li> Sterilize	the	loop,	wait	for	it	to	cool	and	streak	again	starting	from	the	end	of	the	first	line.	This	will
-spread	out	the	bacteria	further.</li>
-<li>Repeat	previous	step	starting	at	the	end	of	the	second	zig-zag	line.	This	will	spread	out	the	bacteria
-even	more.</li>
-<li>Repeat	again,	as	needed.</li>
-<li>Incubate	plates	at	37	°C	overnight	(12	- 14	hours)	or	at	room	temperature	for 24	hours.	Make	sure
-plates	are	incubated	with	agar	layer	on	top	(and	lid	on	the	bottom).</li>
-<li>Plates	with	colonies	can	be	stored	at	4	°C	(fridge)	and	can	be	used	for	at	least	a	couple	of	weeks.
-Make	sure	plates	are	sealed	with	parafilm,	labeled	and	oriented	correctly	(agar	on	top)	while	stored
-in	the	fridge.</li>
-</ol>
-<h2>Transformation</h2>
-<p> A process in which we stress competent cells in a series of times heat shocks and ice baths. This allows the cells to take in the plasmids we want them to have. </p>
-<h4>Materials</h4>
-<ul style="list-style-type:disc;">
-<li>100 μL aliquots of competent cells</li>
-<li>Ice block</li>
-<li>DNA ligation mix</li>
-<li>Micropipettes and sterile pipette tips</li>
-<li>Glass beads</li>
-<li>Control plasmid DNA </li>
-<li>Water bath at 42 °C (or 37 °C) </li>
-<li>Luria broth</li>
-<li> Incubator 37 °C with a rotisserie </li>
-<li> Luria Broth agar plates (with desired antibiotic) </li>
-<li>Heat block</li>
-<h4>Protocol</h4>
-<ol>
-<li> Obtain competent cells from a -80°C freezer and let it thaw on ice for 5 minutes . </li>
-<li> After labeling each tube, use a sterile tip to add 5 μL of the ligation mix to the competent cells and leave them on ice for one hour. </li>
-<li> Using a Heatblock for 45 sec. at 42 degrees C. (Remember to turn off the heatblock.) </li>
-<li> Immediately after heat schocking the cells, place them back on ice for 5 minutes. </li>
-<li> Using a sterile tip add  950 μL Luria broth to the cells and parafilm the tubes </li>
-<li> Place the parafilmed tubes into a rotisserie for 1 hour. </li>
-<li> Once the tubes are done incubating take off the parafilm. </li>
-<li> Warm the Luria broth agar plates with the desired antibiotic resitstance at room temperature. </li>
-<li> Label the plates with the desired product, name, and date. </li>
-<li> Plate 100 μL of the transformed cells in to seperate plates. </li>
-<li> Place 3 to 5 glass beads into the plate to spread the cells evenly.</li>
-<li> Shake the plates in an up and down motion for 2 minutes.</li>
-<li> Remove beads and dispose of them into a waste bin.</li>
-<li>Parafilm the plates and place in the rotisserie at 37°C for 16 hours. </li>
-</ol>
-</div>
+</div></div><h2>Materials</h2><div class="materials-editor" data-editor="materials"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><ul class="materials"><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m1134" data-index="0">DNA <b>1 µg</b></div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m1135" data-index="1"><a href="https://www.neb.com/tools-and-resources/usage-guidelines/nebuffer-performance-chart-with-restriction-enzymes" rel="nofollow noopener noreferrer" target="_blank">NEBuffer</a></div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c1136" data-index="1,0">1X</div></li></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m1137" data-index="2"><a href="https://www.neb.com/products/restriction-endonucleases/restriction-endonucleases?&amp;gclid=CLjO8djMx80CFYRrfgodvKsNkw" rel="nofollow noopener noreferrer" target="_blank">NEB Restriction Enzymes</a></div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m1138" data-index="3">Deionized Water</div><ul class="components"></ul></li></ul></div><h2>Procedure</h2><div class="stepsEditor mediocre"><div class="editor" data-editor="steps"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="mediocre-item is-header" data-scroll-id="s1139"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1139" data-index="0">Sequential Double Digest</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1139" data-index="0" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step" data-scroll-id="s1140"><ol class="steps"><li value="1"><div class="step-checkbox"><span class="glyphicon glyphicon-ok" icon="ok"></span></div><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1140" data-index="1">Set up the following reaction using the restriction endonuclease that has the lowest salt concentration in its recommended buffer (total reaction volume <b>50 µl</b>).</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1140" data-index="1" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s1141"><ol class="steps"><li value="2"><div class="step-checkbox"><span class="glyphicon glyphicon-ok" icon="ok"></span></div><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1141" data-index="2">Set up the following digest reaction on ice</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1141" data-index="2" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-table" data-scale-for-print="print1808" data-scroll-id="s1142"><div class="mediocre-tableEditable printable-container-scaleForPrint" data-formattable="true" data-index="3" data-printable-layout-width="410" data-subindex="cell"><div></div><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="hiddenFocusEditable editable hiddenFocusEditable--fullTable" contenteditable="true" data-cid="s1142-table" data-formattable="true" data-index="3" data-subindex="table"></div><div class="u-position-relative mediocre-tableEditable-scrollAreaWrapper" style="margin-left: 0px; margin-right: 0px;"><div class="mediocre-tableEditable-scrollArea" style="padding-top: 47px;"><div class="mediocre-tableEditable-tableWrapper" style="padding-left: 0px;"><div class="mediocre-tableEditable-border multi-editable" style="left: -6px; top: -43px; bottom: 4px; width: 402px; min-width: 12px;"></div><div class="u-position-relative u-flex-display"><table class="mediocre-tableEditable-table mediocre-tableEditable-table--main" style="width: 390px;"><colgroup><col style="width: 45px;"></col><col data-id="0" style="width: 145px;"></col><col data-id="1" style="width: 100px;"></col><col data-id="2" style="width: 100px;"></col></colgroup><tbody><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--header"></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible mediocre-tableEditable-axisCell--header">A</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><div class="u-flex-display"></div></div></div></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible mediocre-tableEditable-axisCell--header">B</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><div class="u-flex-display"></div></div></div></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible mediocre-tableEditable-axisCell--header">C</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><div class="u-flex-display"></div></div></div></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">1</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><div class="u-flex-display"></div></div></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s1142-0-0" data-namespace="table-ns1718"><span></span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s1142-0-1" data-namespace="table-ns1718"><span>Volume (µl)</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s1142-0-2" data-namespace="table-ns1718"><span></span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">2</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><div class="u-flex-display"></div></div></div></td><td class="cell is-text is-bold is-alignLeft cell-contents" data-cid="table-s1142-1-0" data-namespace="table-ns1718"><span>Buffer (10x)</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-s1142-1-1" data-namespace="table-ns1718"><span>5</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s1142-1-2" data-namespace="table-ns1718"><span></span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">3</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><div class="u-flex-display"></div></div></div></td><td class="cell is-text is-bold is-alignLeft cell-contents" data-cid="table-s1142-2-0" data-namespace="table-ns1718"><span>DNA *</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s1142-2-1" data-namespace="table-ns1718" style="color: rgb(0, 0, 0); background-color: rgb(79, 203, 125);"><span>Input Volume for ng</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s1142-2-2" data-namespace="table-ns1718"><span></span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">4</div><div class="u-flex-display invisible"></div></div></td><td class="cell is-text is-bold is-alignLeft cell-contents" data-cid="table-s1142-3-0" data-namespace="table-ns1718"><span>Restriction Enzyme #1 **</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-s1142-3-1" data-namespace="table-ns1718"><span>1</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s1142-3-2" data-namespace="table-ns1718"><span></span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">5</div><div class="u-flex-display invisible"></div></div></td><td class="cell is-text is-bold is-alignLeft cell-contents" data-cid="table-s1142-4-0" data-namespace="table-ns1718"><span>Deionized Water (μl)</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-s1142-4-1" data-namespace="table-ns1718"><span>1</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s1142-4-2" data-namespace="table-ns1718"><span></span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="errorTriangle aggregateValidationIndicator errorTriangle--illegal" data-original-title="" title=""></div><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">6</div><div class="u-flex-display invisible"></div></div></td><td class="cell is-text is-bold is-alignLeft cell-contents" data-cid="table-s1142-5-0" data-namespace="table-ns1718"><span>Total Volume (μl)</span></td><td class="cell is-text is-alignLeft cell--error" data-cid="table-s1142-5-1" data-namespace="table-ns1718"><div class="cell-innerWrapper"><div class="errorTriangle cellValidationIndicator errorTriangle--illegal" data-original-title="" title=""></div><div class="cell-contents"><span>#VALUE!</span></div></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s1142-5-2" data-namespace="table-ns1718"><span></span></td></tr></tbody><tfoot></tfoot></table><div class="mediocre-tableEditable-fillerTableWrapper" style="margin-right: 10px;"><table class="mediocre-tableEditable-fillerTable" style="display: none;"><tbody><tr style="height: 0px;"><td></td></tr><tr style="height: 0px;"><td></td></tr><tr style="height: 0px;"><td></td></tr><tr style="height: 0px;"><td></td></tr><tr style="height: 0px;"><td></td></tr><tr style="height: 0px;"><td></td></tr><tr style="height: 0px;"><td></td></tr></tbody></table></div></div></div></div></div><div class="mediocre-tableEditable-titleBarWrapper"><div class="mediocre-tableEditable-titleBar" style="height: 37px; padding-left: 0px;"><div class="mediocre-tableEditable-tableName u-pointer"><div class="u-flex-grow u-overflow-hidden u-verticalCenterItems"><div class="u-ellipsis-text"><span class="u-ellipsis-text">Table1</span></div></div><div class="u-flex-none"></div></div><div class="u-flex-none u-tiny-spacing-left u-verticalCenterItems table-floater"><svg class="svgIcon workflowTable-tableCollapser u-pointer"><use xlink:href="#chevron-up_c0f0f77c91b0d9072b0d8255f9f54e3c"></use></svg></div></div></div></div></div><div class="mediocre-item is-description" data-scroll-id="s1143"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1143" data-index="4">*A 50 µl reaction volume is recommended for digestion of 1 µg of substrate.</div></div></li></ul></div><div class="mediocre-item is-description" data-scroll-id="s1144"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1144" data-index="5">** Restriction Enzyme,  10 units is sufficient, generally <b>1 µl</b> is used</div></div></li></ul></div><div class="mediocre-item is-description" data-scroll-id="s1145"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1145" data-index="6">***The enzyme should be the last component added to reaction</div></div></li></ul></div><div class="mediocre-item is-step" data-scroll-id="s1146"><ol class="steps"><li value="3"><div class="step-checkbox"><span class="glyphicon glyphicon-ok" icon="ok"></span></div><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1146" data-index="7">Mix components by pipetting the reaction mixture up and down, or by "flicking" the reaction tube.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1146" data-index="7" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s1147"><ol class="steps"><li value="4"><div class="step-checkbox"><span class="glyphicon glyphicon-ok" icon="ok"></span></div><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1147" data-index="8">Quick ("touch") spin-down in a microcentrifuge. Do not vortex the reaction.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1147" data-index="8" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s1148"><ol class="steps"><li value="5"><div class="step-checkbox"><span class="glyphicon glyphicon-ok" icon="ok"></span></div><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1148" data-index="9">Incubate for 1 hour at the enzyme-specific appropriate temperature.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1148" data-index="9" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-timer" data-scroll-id="s1149"><div class="timer-outer"><div><div class="timer-editable-wrapper"><span><span class="step-timer inline-step-timer"><div class="time-display">01:00<span class="time-small">:00</span></div><div class="time-controls"><a><span class="glyphicon glyphicon-play" icon="play"></span></a></div></span></span></div></div></div></div><div class="mediocre-item is-description" data-scroll-id="s1150"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1150" data-index="11">Can be decreased to 5-15 minutes by using a <a href="https://www.neb.com/products/restriction-endonucleases/hf-nicking-master-mix-time-saver-other/time-saver-qualified-restriction-enzymes/time-saver-qualified-restriction-enzymes" rel="nofollow noopener noreferrer" target="_blank">Time-Saver™ Qualified Restriction Enzyme</a></div></div></li></ul></div><div class="mediocre-item is-description" data-scroll-id="s1151"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1151" data-index="12">See the <a href="https://www.neb.com/tools-and-resources/usage-guidelines/nebuffer-performance-chart-with-restriction-enzymes" rel="nofollow noopener noreferrer" target="_blank">NEBuffer Activity/Performance Chart with Restriction Enzymes</a> for the incubation temperatures.</div></div></li></ul></div><div class="mediocre-item is-step" data-scroll-id="s1152"><ol class="steps"><li value="6"><div class="step-checkbox"><span class="glyphicon glyphicon-ok" icon="ok"></span></div><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1152" data-index="13">Adjust the salt concentration of the reaction (using a small volume of a concentrated salt solution) to approximate the reaction conditions of the second restriction endonuclease. </div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1152" data-index="13" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description" data-scroll-id="s1153"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1153" data-index="14">Alternatively, a spin column can be used to isolate the DNA prior to the second reaction.</div></div></li></ul></div><div class="mediocre-item is-step" data-scroll-id="s1154"><ol class="steps"><li value="7"><div class="step-checkbox"><span class="glyphicon glyphicon-ok" icon="ok"></span></div><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1154" data-index="15">Add the second enzyme.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1154" data-index="15" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description" data-scroll-id="s1155"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1155" data-index="16">** Restriction Enzyme #2,  10 units is sufficient, generally <b>1 µl</b> is used</div></div></li></ul></div><div class="mediocre-item is-step" data-scroll-id="s1156"><ol class="steps"><li value="8"><div class="step-checkbox"><span class="glyphicon glyphicon-ok" icon="ok"></span></div><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1156" data-index="17">Mix components by pipetting the reaction mixture up and down, or by "flicking" the reaction tube.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1156" data-index="17" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s1157"><ol class="steps"><li value="9"><div class="step-checkbox"><span class="glyphicon glyphicon-ok" icon="ok"></span></div><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1157" data-index="18">Quick ("touch") spin-down in a microcentrifuge. Do not vortex the reaction.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1157" data-index="18" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s1158"><ol class="steps"><li value="10"><div class="step-checkbox"><span class="glyphicon glyphicon-ok" icon="ok"></span></div><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1158" data-index="19">Incubate for 1 hour at the enzyme-specific appropriate temperature.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1158" data-index="19" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description" data-scroll-id="s1159"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1159" data-index="20">Can be decreased to 5-15 minutes by using a <a href="https://www.neb.com/products/restriction-endonucleases/hf-nicking-master-mix-time-saver-other/time-saver-qualified-restriction-enzymes/time-saver-qualified-restriction-enzymes" rel="nofollow noopener noreferrer" target="_blank">Time-Saver™ Qualified Restriction Enzyme</a></div></div></li></ul></div><div class="mediocre-item is-description" data-scroll-id="s1160"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1160" data-index="21">See the <a href="https://www.neb.com/tools-and-resources/usage-guidelines/nebuffer-performance-chart-with-restriction-enzymes" rel="nofollow noopener noreferrer" target="_blank">NEBuffer Activity/Performance Chart with Restriction Enzymes</a> for the incubation temperatures.</div></div></li></ul></div></div></div></div></div></div></div></div></div><div class="printable-page-container"><div class="protocolEditor"><div></div><div class="protocolEditor-pageContainer"><div class="protocolEditor-page"><div class="protocolEditor-contentWrapper"><h1 class="mediocre-titleEditor is-locked"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="false" data-cid="ep1180-title" data-index="0">Ligation Protocol WITH T4 DNA Ligase (M0202)</div></h1><div><h2>Introduction</h2><div class="intro-editor" data-editor="intro"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="true" data-cid="ep1180-intro" data-index="0"><i>Please see the </i><a href="https://www.neb.com/protocols/1/01/01/dna-ligation-with-t4-dna-ligase-m0202" rel="nofollow noopener noreferrer" target="_blank"><i>NEB website</i></a><i> for supporting information on this protocol.</i>
+</div></div><h2>Materials</h2><div class="materials-editor" data-editor="materials"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><ul class="materials"><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m1163" data-index="0"><a href="https://www.neb.com/products/b0202-t4-dna-ligase-reaction-buffer" rel="nofollow noopener noreferrer" target="_blank">10X T4 DNA Ligase Reaction Buffer</a></div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m1164" data-index="1"><a href="https://www.neb.com/products/m0202-t4-dna-ligase" rel="nofollow noopener noreferrer" target="_blank">T4 DNA Ligase</a></div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m1165" data-index="2">Vector DNA (4kb)</div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m1166" data-index="3">Insert DNA (1kb)</div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m1167" data-index="4">Nuclease-free water</div><ul class="components"></ul></li></ul></div><h2>Procedure</h2><div class="stepsEditor mediocre"><div class="editor" data-editor="steps"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="mediocre-item is-header" data-scroll-id="s1168"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1168" data-index="0">Set up the T4 DNA Ligase Reaction</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1168" data-index="0" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-description" data-scroll-id="s1169"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1169" data-index="1"><i>Note: </i>T4 DNA Ligase should be added last. The table shows a ligation using a molar ratio of 1:3 vector to insert for the indicated DNA sizes. <i> Use </i><a href="http://nebiocalculator.neb.com/#!/" rel="nofollow noopener noreferrer" target="_blank"><i>NEB calculator</i></a><i> to calculate molar ratios.</i></div></div></li></ul></div><div class="mediocre-item is-step" data-scroll-id="s1170"><ol class="steps"><li value="1"><div class="step-checkbox"><span class="glyphicon glyphicon-ok" icon="ok"></span></div><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1170" data-index="2">Thaw the T4 DNA Ligase Buffer and resuspend at room temperature.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1170" data-index="2" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description" data-scroll-id="s1171"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1171" data-index="3"><i>Tip: Alicuote the 10x buffer less concentrated so when thawing, the DTT gets soluble more easily.</i></div></div></li></ul></div><div class="mediocre-item is-step" data-scroll-id="s1172"><ol class="steps"><li value="2"><div class="step-checkbox"><span class="glyphicon glyphicon-ok" icon="ok"></span></div><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1172" data-index="4">Set up the following reaction in a microcentrifuge tube on ice:</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1172" data-index="4" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-table" data-scale-for-print="print1809" data-scroll-id="s1173"><div class="mediocre-tableEditable printable-container-scaleForPrint" data-formattable="true" data-index="5" data-printable-layout-width="416" data-subindex="cell"><div></div><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="hiddenFocusEditable editable hiddenFocusEditable--fullTable" contenteditable="true" data-cid="s1173-table" data-formattable="true" data-index="5" data-subindex="table"></div><div class="u-position-relative mediocre-tableEditable-scrollAreaWrapper" style="margin-left: 0px; margin-right: 0px;"><div class="mediocre-tableEditable-scrollArea" style="padding-top: 47px;"><div class="mediocre-tableEditable-tableWrapper" style="padding-left: 0px;"><div class="mediocre-tableEditable-border multi-editable" style="left: -6px; top: -43px; bottom: 4px; width: 408px; min-width: 12px;"></div><div class="u-position-relative u-flex-display"><table class="mediocre-tableEditable-table mediocre-tableEditable-table--main header-row" style="width: 396px;"><colgroup><col style="width: 45px;"></col><col data-id="0" style="width: 200px;"></col><col data-id="1" style="width: 151px;"></col></colgroup><tbody><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--header"></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible mediocre-tableEditable-axisCell--header">A</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><div class="u-flex-display"></div></div></div></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible mediocre-tableEditable-axisCell--header">B</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><div class="u-flex-display"></div></div></div></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">1</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><div class="u-flex-display"></div></div></div></td><td class="cell is-text is-bold is-alignLeft header-cell cell-contents" data-cid="table-s1173-0-0" data-namespace="table-ns1724"><span>Component</span></td><td class="cell is-text is-bold is-alignLeft header-cell cell-contents" data-cid="table-s1173-0-1" data-namespace="table-ns1724"><span>Volume (μl)</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">2</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><div class="u-flex-display"></div></div></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s1173-1-0" data-namespace="table-ns1724"><span>10X T4 DNA Ligase Buffer</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-s1173-1-1" data-namespace="table-ns1724"><span>2</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">3</div><div class="u-flex-display invisible"></div></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s1173-2-0" data-namespace="table-ns1724"><span>Vector DNA: 50 ng (0.020 pmol)</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s1173-2-1" data-namespace="table-ns1724"><span></span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">4</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><div class="u-flex-display"></div></div></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s1173-3-0" data-namespace="table-ns1724"><span>Insert DNA: 37.5 ng (0.060 pmol)</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s1173-3-1" data-namespace="table-ns1724"><span></span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">5</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><div class="u-flex-display"></div></div></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s1173-4-0" data-namespace="table-ns1724"><span>Nuclease-free water</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-s1173-4-1" data-namespace="table-ns1724"><span>17</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">6</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><div class="u-flex-display"></div></div></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-s1173-5-0" data-namespace="table-ns1724"><span>T4 DNA Ligase</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-s1173-5-1" data-namespace="table-ns1724"><span>1</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-labelWrapper"><div class="mediocre-tableEditable-axisCell-labelSizer u-overflow-wrap-break-word invisible">7</div><div class="u-flex-display invisible"></div><div class="mediocre-tableEditable-axisCell-label u-verticalCenterItems"><div class="u-flex-display"></div></div></div></td><td class="cell is-text is-underline is-alignLeft cell-contents" data-cid="table-s1173-6-0" data-namespace="table-ns1724"><span>Total</span></td><td class="cell is-value is-underline is-alignRight cell-contents" data-cid="table-s1173-6-1" data-namespace="table-ns1724"><span>20</span></td></tr></tbody><tfoot></tfoot></table><div class="mediocre-tableEditable-fillerTableWrapper" style="margin-right: 10px;"><table class="mediocre-tableEditable-fillerTable" style="display: none;"><tbody><tr style="height: 0px;"><td></td></tr><tr style="height: 0px;"><td></td></tr><tr style="height: 0px;"><td></td></tr><tr style="height: 0px;"><td></td></tr><tr style="height: 0px;"><td></td></tr><tr style="height: 0px;"><td></td></tr><tr style="height: 0px;"><td></td></tr><tr style="height: 0px;"><td></td></tr></tbody></table></div></div></div></div></div><div class="mediocre-tableEditable-titleBarWrapper"><div class="mediocre-tableEditable-titleBar" style="height: 37px; padding-left: 0px;"><div class="mediocre-tableEditable-tableName u-pointer"><div class="u-flex-grow u-overflow-hidden u-verticalCenterItems"><div class="u-ellipsis-text"><span class="u-ellipsis-text">Table1</span></div></div><div class="u-flex-none"></div></div><div class="u-flex-none u-tiny-spacing-left u-verticalCenterItems table-floater"><svg class="svgIcon workflowTable-tableCollapser u-pointer"><use xlink:href="#chevron-up_c0f0f77c91b0d9072b0d8255f9f54e3c"></use></svg></div></div></div></div></div><div class="mediocre-item is-step" data-scroll-id="s1174"><ol class="steps"><li value="3"><div class="step-checkbox"><span class="glyphicon glyphicon-ok" icon="ok"></span></div><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1174" data-index="6">Gently mix the reaction by pipetting up and down and microfuge briefly.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1174" data-index="6" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s1175"><ol class="steps"><li value="4"><div class="step-checkbox"><span class="glyphicon glyphicon-ok" icon="ok"></span></div><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1175" data-index="7">For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes. For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1175" data-index="7" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s1176"><ol class="steps"><li value="5"><div class="step-checkbox"><span class="glyphicon glyphicon-ok" icon="ok"></span></div><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1176" data-index="8">Heat inactivate at 65 degrees C for 10 minutes.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1176" data-index="8" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-timer" data-scroll-id="s1177"><div class="timer-outer"><div><div class="timer-editable-wrapper"><span><span class="step-timer inline-step-timer"><div class="time-display">00:10<span class="time-small">:00</span></div><div class="time-controls"><a><span class="glyphicon glyphicon-play" icon="play"></span></a></div></span></span></div></div></div></div><div class="mediocre-item is-step" data-scroll-id="s1178"><ol class="steps"><li value="6"><div class="step-checkbox"><span class="glyphicon glyphicon-ok" icon="ok"></span></div><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1178" data-index="10">Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1178" data-index="10" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description" data-scroll-id="s1179"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1179" data-index="11">Use 25 uL DH5α cells, and add 2 uL of reaction mixture.</div></div></li></ul></div></div></div></div></div></div></div></div></div><div class="printable-page-container"><div class="protocolEditor"><div></div><div class="protocolEditor-pageContainer"><div class="protocolEditor-page"><div class="protocolEditor-contentWrapper"><h1 class="mediocre-titleEditor is-locked"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="false" data-cid="ep1209-title" data-index="0">Transformation</div></h1><div><h2>Introduction</h2><div class="intro-editor" data-editor="intro"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="true" data-cid="ep1209-intro" data-index="0">A process in which we stress competent cells in a series of times heat shocks and ice baths. This allows the cells to take in the plasmids we want them to have.
+</div></div><h2>Materials</h2><div class="materials-editor" data-editor="materials"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><ul class="materials"><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m1182" data-index="0">100 μL aliquots of competent cells</div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m1183" data-index="1">ice block</div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m1184" data-index="2">DNA ligation mix</div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m1185" data-index="3">micropipettes and sterile pipette tips</div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m1186" data-index="4">Glass beads</div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m1187" data-index="5">control plasmid DNA</div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m1188" data-index="6">water bath at 42 °C (or 37 °C)</div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m1189" data-index="7">Luria Broth</div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m1190" data-index="8">Incubator 37 °C with a rotisserie</div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m1191" data-index="9">Luria Broth agar plates (with desired antibiotic)</div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m1192" data-index="10">Heatblock</div></li></ul></div><h2>Procedure</h2><div class="stepsEditor mediocre"><div class="editor" data-editor="steps"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="mediocre-item is-header" data-scroll-id="s1194"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1194" data-index="0">Transformation Protocol</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1194" data-index="0" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step" data-scroll-id="s1195"><ol class="steps"><li value="1"><div class="step-checkbox"><span class="glyphicon glyphicon-ok" icon="ok"></span></div><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1195" data-index="1">Obtain competent cells from a -80°C freezer and let it thaw on ice for 5 minutes .</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1195" data-index="1" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s1196"><ol class="steps"><li value="2"><div class="step-checkbox"><span class="glyphicon glyphicon-ok" icon="ok"></span></div><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1196" data-index="2">After labeling each tube, use a sterile tip to add 5 μL of the ligation mix to the competent cells and leave them on ice for one hour.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1196" data-index="2" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s1197"><ol class="steps"><li value="3"><div class="step-checkbox"><span class="glyphicon glyphicon-ok" icon="ok"></span></div><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1197" data-index="3">Using a Heatblock for 45 sec. at 42 degrees C. (Remember to turn off the heatblock.)</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1197" data-index="3" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s1198"><ol class="steps"><li value="4"><div class="step-checkbox"><span class="glyphicon glyphicon-ok" icon="ok"></span></div><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1198" data-index="4">Immediately after heat schocking the cells, place them back on ice for 5 minutes.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1198" data-index="4" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s1199"><ol class="steps"><li value="5"><div class="step-checkbox"><span class="glyphicon glyphicon-ok" icon="ok"></span></div><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1199" data-index="5">Using a sterile tip add  950 μL <span style="color:#222222"><span style="background-color:#FFFFFF">Luria broth</span></span> to the cells and parafilm the tubes</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1199" data-index="5" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s1200"><ol class="steps"><li value="6"><div class="step-checkbox"><span class="glyphicon glyphicon-ok" icon="ok"></span></div><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1200" data-index="6">Place the parafilmed tubes into a rotisserie for 1 hour.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1200" data-index="6" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s1201"><ol class="steps"><li value="7"><div class="step-checkbox"><span class="glyphicon glyphicon-ok" icon="ok"></span></div><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1201" data-index="7">Once the tubes are done incubating take off the parafilm.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1201" data-index="7" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s1202"><ol class="steps"><li value="8"><div class="step-checkbox"><span class="glyphicon glyphicon-ok" icon="ok"></span></div><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1202" data-index="8">Warm the <span style="color:#222222"><span style="background-color:#FFFFFF">Luria broth</span></span> agar plates with the desired antibiotic resitstance at room temperature.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1202" data-index="8" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s1203"><ol class="steps"><li value="9"><div class="step-checkbox"><span class="glyphicon glyphicon-ok" icon="ok"></span></div><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1203" data-index="9">Label the plates with the desired product, name, and date.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1203" data-index="9" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s1204"><ol class="steps"><li value="10"><div class="step-checkbox"><span class="glyphicon glyphicon-ok" icon="ok"></span></div><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1204" data-index="10">Plate 100 <span style="color:#344043"><span style="background-color:#FFFFFF">μL</span></span> of the transformed cells in to seperate plates.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1204" data-index="10" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s1205"><ol class="steps"><li value="11"><div class="step-checkbox"><span class="glyphicon glyphicon-ok" icon="ok"></span></div><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1205" data-index="11">Place 3 to 5 glass beads into the plate to spread the cells evenly.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1205" data-index="11" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s1206"><ol class="steps"><li value="12"><div class="step-checkbox"><span class="glyphicon glyphicon-ok" icon="ok"></span></div><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1206" data-index="12">Shake the plates in an up and down motion for 2 minutes.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1206" data-index="12" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s1207"><ol class="steps"><li value="13"><div class="step-checkbox"><span class="glyphicon glyphicon-ok" icon="ok"></span></div><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1207" data-index="13"> Remove beads and dispose of them into a waste bin.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1207" data-index="13" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s1208"><ol class="steps"><li value="14"><div class="step-checkbox"><span class="glyphicon glyphicon-ok" icon="ok"></span></div><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1208" data-index="14">Parafilm the plates and place in the rotisserie at 37°C for 16 hours. </div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1208" data-index="14" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div></div></div></div></div></div></div></div></div></div></div></div></div></div></div>
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Difference between revisions of "Team:Bio Without Borders/Notebook"

Revision as of 17:35, 30 September 2019

BwoB iGEM 2019

iGEM_Meeting_Notebook.pdf

Sequential Double Digest

Introduction

Materials

Procedure

Ligation Protocol WITH T4 DNA Ligase (M0202)

Introduction

Materials

Procedure

Transformation

Introduction

Materials

Procedure