Team:CMUQ/Notebook

iGEM CMUQ 2019

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Team Members Collaborations Sponsors Attributions Project Description Details Hardware Software Demonstrate Model Results Lab Notebook Parts Overview Basic Parts Improved Parts Composite Parts Human Practices Safety Outreach Awards Hardware Software Entrepreneurship Model Measurement Characterization Judging Form
Lab Notebook

Lab Notebook

Mini-prep pY016 Plasmid
Wednesday 15 May, 2019

(To obtain plasmid from DH5-α saturated culture - adapted from Qiagen Spin Mini Prep)

Procedure:

  1. Pellet 5 ml bacterial overnight culture by centrifugation at > 8000 rpm (6800 x gl for3 min at room temperature15-25 C).
  2. Completely resuspend pelleted bacterial cells in 250 ul Buller PI and transfer to a microcentrifuge tube. Ensure that no dups of cells remain.
  3. Add 250 ul Buffer P2 and mix by inverting the tube 10-12 times until the solution turns completely blue. Incubate for 5 min. Do not allow lysis to proceed for for more than 5 mins.
  4. Add 350 ul Buffer N3 and mix immediately and thoroughly by inverting the tube 10-12 times until the solution turns colorless
  5. Centrifuge tor 10 min.
  6. Apply the supernatant from step 5 to the QIAprep spin column by decanting or pipetting. Centrifuge for 30 – 60 s and discard the flow-through
  7. Wash the QIAprep spin column by adding 500 ul Buffer PB. Centrifuge as in step 6
  8. Wash the QIAprep spin column by adding 750 ul Buffer PE. Centrifuge as in step 6. Transfer the QIAprep spin column to the collection tube.
  9. Centrifuge for 1 min to remove residual wash buffer.


Validation:

0.6% agarose gel was run to check for quality of extracted plasmid. 8μl of sample was added to 2μl of 6x loading dye.

Results:

Amplification of HBB_WT and HBB_C Templates
Wednesday 15 May, 2019

PCR Reagents:

ReagentVolume (μl)
Nuclease-free water 31.5
10x PCR buffer + 15mM MgCl2 5
10mM dNTPs 1
Prefix primer (10μM) 1
SuffixR primer (10μM) 1
gBLOCK DNA (10ng/μl) 10
Ampli taq gold (2.5U/μl) 0.5
Total 50


PCR Conditions (Dsp-T7):

StepTemperature (°C)DurationNo. of Cycles
Initial Denaturation 95 10 min 1
Denaturation 95 15 sec 30
Annealing 55 30 sec 30
Extension 72 1 min 30
Finish 72 5 min 1
Hold 4 ∞ -


Results:

Transformation of pY016 Plasmid into BL21 Cells for Protein Expression
Thursday 16 May, 2019

Materials:

  • 17 × 100mm polypropylene culture tubes
  • Competent BL21 cells
  • 42°C water bath
  • SOC medium
  • LB+AMP+CHL agar plates


Procedure:

  1. Add 1μl of purified vector/plasmid to glass tube. Tap gently (DO NOT MIX).
  2. Incubate on ice for 10 mins.
  3. Incubate at 42°C for 45-50 secs - heat shock step.
  4. Place on ice for 2 mins.
  5. Add 900μl of cold SOC media.
  6. Shake for 1hr at 37°C.
  7. Spread plate 100μl on LB+AMP+CHL plates.


Results:

Transformants were not observed on plate, only a single colony was obseved and was suspected to be contamination.

Transformation of pY016 Plasmid into DH5-α Cells for Plasmid Replication
Thursday 16 May, 2019

Materials:

  • 37°C shaking and non-shaking incubator
  • LB+AMP
  • SOC medium on ice
  • 42°C water bath
  • 17 × 100mm polypropylene culture tubes
  • Competent DH5-α cells


Procedure:

  1. Add 1μl of purified vector/plasmid to glass tube. Tap gently (DO NOT MIX).
  2. Incubate on ice for 30 mins.
  3. Incubate at 42°C for 30 secs exactly - heat shock step.
  4. Place on ice.
  5. Add 250μl of warm SOC media.
  6. Shake for 1hr at 37°C.
  7. Spread plate 100μl on LB+AMP plates.


Results:

Transformants were observed on plate, and were inoculated into tubes in order to perform plasmid isolation again.

Inoculation of DH5-α transformants
Sunday 19 May, 2019

Procedure:

  1. Add 5ml of LB+AMP media to sterile glass tubes.
  2. Using an inoculating loop, pick a single colony of transformants, and place it in the glass tube.
  3. Shake loop to ensure the transformant bacteria has transferred to solution.
  4. Keep tubes shaking in the air shaker at 37°C overnight.


Four tubes were prepared from the DH5-α transformants.
Mini-prep pY016 Plasmid
Monday 20 May, 2019

(To obtain plasmid from DH5-α saturated culture)

Procedure:

  1. Pellet 5 ml bacterial overnight culture by centrifugation at > 8000 rpm (6800 x gl for3 min at room temperature 15-25 C).
  2. Completely resuspend pelleted bacterial cells in 250 ul Buller PI and transfer to a microcentrifuge tube. Ensure that no dups of cells remain.
  3. Add 250 ul Buffer P2 and mix by inverting the tube 10-12 times until the solution turns completely blue. Incubate for 5 min. Do not allow lysis to proceed for for more than 5 mins.
  4. Add 350 ul Buffer N3 and mix immediately and thoroughly by inverting the tube 10-12 times until the solution turns colorless
  5. Centrifuge tor 10 min.
  6. Apply the supernatant from step 5 to the QIAprep spin column by decanting or pipetting. Centrifuge for 30 – 60 s and discard the flow-through
  7. Wash the QIAprep spin column by adding 500 ul Buffer PB. Centrifuge as in step 6
  8. Wash the QIAprep spin column by adding 750 ul Buffer PE. Centrifuge as in step 6. Transfer the QIAprep spin column to the collection tube.
  9. Centrifuge for 1 min to remove residual wash buffer.


Validation:

0.6% agarose gel was run to check for quality of extracted plasmid. 8μl of sample was added to 2μl of 6x loading dye.
Agarose Gel Electrophoresis to Check pY016 Plasmid Extraction
Monday 20 May, 2019

Procedure:

Preparing the agarose gel

  1. The fragments generated by digestion of the DNA will be separated by electrophoresis through agarose gels. Regular agarose melts at 100˚C but solidifies at about 42˚C. So we melt the agarose at higher temperature in water or buffer, pour it into a form, and allow it to solidify by simply letting it cool below 42˚C. The gel is formed and run as a horizontal slab since it does noy have enough structural strength to be used vertically. Before the gel material solidifies, there will be wells into which the samples can be introduced.
  2. The volume of sample that will be loaded in each sample well is not large (15-25µl) so the wells need not be very deep. The level of the comb is set so that there will be some agarose below the teeth of the comb to keep the sample from leaking onto the gel platform. Make any adjustments in the height of the comb before you start pouring the gel.
  3. Weigh 0.35g agarose and add 45ml distilled water in a conical flask. Microwave until the agarose dissolves. You must add 5ml of the 10X TBE buffer found on the benchtop.This will give you 50ml of agarose (final concentration 0.7%) in 1X buffer. Let the gel cool a bit
  4. CAUTION! ETHIDIUM BROMIDE IS TOXIC! WEAR GLOVES AND SAFETY GOGGLES
  5. Go to the hood, and the instructor will add 10µl of ethidium bromide (5mg/ml) to the bottle of prepared agarose. Mix thoroughly. This is enough solution to pour a gel in the gel apparatus. Each gel can hold a total of 10 samples, depending on the type and number of combs used.


Pouring the gel:

Set up the gel apparatus as was demonstrated to you. Place the comb onto the gel tray. Pour the gel onto the tray until the wells of the comb are submerged. Pipet off any bubbles. When the gel has polymerized (it become turbid), very gently pull out the comb. Immediately flood the top of the gel and wells with 1XTBE (you will need to make about 500ml per gel)

Preparing and Loading the Samples:

  1. The samples of plasmid DNA are simply 5 ml of each DNA plus 2 ml of 10X tracking dye (final concentration = 1X). Pipet these volumes into 1.7ml microcentrifuge tubes and centrifuge briefly to mix them together. Keep the samples chilled until you are ready to load them.
  2. We will load the sample wells and run the gel while it is submerged in buffer (although the illustration to the left does not show a submerged gel). The tracking dye solution contains glycerol or a dense polymer, so the final sample will be somewhat denser than the buffer that fills the sample well. Therefore, the samples pass through the buffer and lay in the bottom of the well.


Lane Sample

Attach the gel apparatus to a power supply in the proper orientation and adjust the voltage to 100 volts. After 30 minutes turn off the power and disconnect the leads.

Photographing the gel

Wearing gloves, lift the tray out of the buffer very carefully - the gel is likely to slide off the tray. Transfer the gel (or tray and gel) to a gel box and take it to the short wavelength ultra-violet light box. Illumination of the ethidium bromide by ultra-violet light causes fluorescence in the visible wavelengths; you CAN see DNA as orange bands. However, since the ultra-violet light is hazardous to your eyes, we urge you to take a photograph of the gel rather than examine it visually - you can study the photo all you like with no risks!

Capturing and Saving Images with the UV light box

Place the tray with the gel in the UV light box, and select UV light. Adjust the Aperature, Zoom. Take photo and save on the Thumb drive.
Agarose Gel Electrophoresis to Check Digest Product
Tuesday 21 May, 2019

A 0.6% Agarose gel was prepared and run at 100V for 60 minutes.
Inoculation of Rosetta and BL21 transformants
Wednesday 22 May, 2019

Procedure:

  1. Add 5ml of LB+AMP+CHL media to sterile glass tubes.
  2. Using an inoculating loop, pick a single colony of transformants, and place it in the glass tube.
  3. Shake loop to ensure the transformant bacteria has transferred to solution.
  4. Keep tubes shaking in the air shaker at 37°C overnight.
  5. Then next day, the culture is diluted in to the flask with 20ml LB+AMP+CHL media, the OD (in Kletts) is meausred to monitor the growth of the cell.
  6. Once the OD reaches 30 Kletts and when we observe the doubling of the OD, 1mM IPTG is added to the culture.
  7. Keep the flask shaking in the air shaker at 37°C for various period of times(4 hours, 12 hours, 18 hours and 22 hours)
  8. Proteins are harvested by centrifuging at 5000 rmp for 5 min.
  9. The quality and quantity of the proteins are veried by running the SDS-PAGE.




Cas12a activity – testing of template and guides – Protocol according to EnGen® Lba Cas12a (Cpf1)
Introduction

In order to test the activity of Cas12a and the design of the gRNAs, a reaction tube was prepared which was then tested with DNAse Alert Substrate and run on the bionalanyzer for more sensitive detection.

Materials:

  • 20 ul of Nuclease Free Water
  • 3 ul of 10X 2.1 Buffer
  • 3 ul of 300 nM guide RNA (Nuclease free water was substituted for control samples with no gRNA)
  • 1 ul of 1µM EnGen® Lba Cas12a (Cpf1) or Elution fraction


Procedure:
(After preparing the reaction tubes)

  1. Pre-incubate tubes for 10min at 25°C
  2. Add 3µl of 30nM template
  3. Mix thoroughly and quick spin
  4. Incubate tubes at 37°C for 10min
  5. Add 1µl Proteinase K (Macharey-Nagel). Mix and pulse-spin
  6. Incubate tubes at 28°C for 10min
  7. 1ul of the reaction mixture was analyzed on the High Sensitivity DNA chip using the Bioanalyser ( Agilent Biotechnologies)and 20µl was analyzed for Cas12a activity using DnaseAlert Substrate (IDT)
Measuring the activity of Ca12a using DNAseAlert Substrate Kit (IDT)
Introduction

To measure the fluorescence emitted due to Cas12a activity, the ssDNA reporter molecule (contaning a quencher and a fluorescent tag) was used were upons its cleavage, Cas12a activity cleavage activity due tocorrect binding is confirmed.

Materials:

  • DNAaseAlert Substrate Kit from IDT
  • Reaction tubes containing: DNA template, gRNA, Cas12a, and buffer


Procedure:

  1. Into each single-use DNaseAlert Substrate tube, 5µl of nuclease-free water was added; followed by 5µl of 10X DNaseAlert buffer. It was mixed up and down
  2. 5µl was added to each well on a clear 96-well plate
  3. 20µl of Cas12a reactions were added
  4. Plate was incubated at 37°C for 1h. To generate a standard curve for enzyme units, varying concentrations of Dnase I (0, 0.0015, 0.015, 0.15kU) (Rnase free DNase I, Cat No: 79254, Qiagen)
  5. Cas12a activity was measured as equivalent units to Dnase activity
  6. Fluorescence was then measured using the HEX channel Ex 536 and Em 556nm using the Varioskan Fluoresence plate reader ( Thermoscientific)