WEEK 1
Monday : 7/15/2019
Learned Basic Lab Techniques
● pipetting
● making agarose gel
● making agar plates with LB + AMP
Agarose Gel Preparation (3%):
1. 1x TAE buffer was prepared by diluting a 50x TAE buffer
2. 1.5 g of Agarose were added to 50 ml of 1x TAE buffer (3%) in a conical flask
3. The solution was placed in a microwave
and mixed until solution became clear
4. 3 microliters of Gel Red were added to the solution
5. A well comb was added to the gel cast, the solution was poured into the gel cast and allowed
to solidify
Cas12 Cleavage with Cra gene:
1. Making 125nM gRNA: 0.2 μl of 1000 μM gRNA and 1.58 ml of water were mixed
2. Making 100nM CRISPR LbCas12a: 1 μl of 100 μM Cas12 and 999 μl of water
3. 1 μl of each solution
were used to make 2 μl 62.5nM gRNA : 50nM Cas12a solution and added to approximately 18 μl of Cra
DNA solution.
4. The same solution was made with 1 μl each of undiluted Cas12a and gRNA solutions (gRNA: 1000
μM; CAS12a: 100 μM).
5. The solutions were left to set for 1.5 hours in 37°C incubator.
6. A Gel electrophoresis was ran for the two samples and the control
Resuspended pcaA, HBcAg and IS481 gBlock gene fragments from IDT
1. For 500ng gBlocks, 50μL of molecular grade water was added to the tube. For 1000ng gBlocks, 100μL of molecular grade water
was added to the tube to reach a final concentration of 10ng/μL
2. The
tubes were vortexed briefly then incubated at 50°C for 15 mins
3. The tubes were briefly vortexed and centrifuged for 5 seconds
4. The concentration of each gBlock resuspension was taken
with the NanoDrop
pcaA: 8.7 ng/μL
HBcAg: 11.2 ng/μL
IS481: 8.2 ng/μL
5. Prepared Thermo Scientific CloneKet PCR Cloning Kit reagent mix in a PCR tube on ice
6. Added 10μL 2X Reaction Buffer
7. Added 1μL resuspended pcaA gBlock gene fragement
8. Added 1μL pJET 1.2/blunt cloning vector (50ng/μL)
9. Added 7μL nuclease-free water
10. Added 1μL T4 DNA Ligase
11. Incubated at room temperature for 5 minutes
12. Repeated
for HBcAg and IS481 gBlocks
Transformation of E.coli via electroporation using ligated pJET vectors incubated for 5 minutes
1. Aliquoted 33μL of electrocompetent E.coli into 3 eppendorf tubes
2. Added either 10μL of pcaA, HBcAg or IS481 ligated pJET vectors into each of 3 eppendorf tubes
3. Pipette
up and down to mix
4. Pipette contents of each eppendorf tube (cells with either pcaA, HBcAg or IS481) into electroporation cuvette
5. Place into electroporation machine and electroporate
6. Resuspend cells in 960μL of SOC by pipetting up and down in cuvette
7. Pour cuvette contents into 3 separate eppendorf tubes
8. Incubated on shaker at 220rpm and 37°C for one hour
9. Centrifuged
for 1 minute at at 13000rpm, discarded 800μL supernatant from each eppendorf tube
10. Pellets were resuspended by pipetting up and down
11. Cells were spread on LB+AMP agar plates and placed
in 37°C incubator overnight
Results of Ligation and Transformation using electroporation and 5 minute ligation incubation:
No growth on plates after overnight incubation.
Tuesday : 7/16/2019
Transformation with Heat Shock and 24 hour ligation incubation
pJET Blunt End Ligation
1. Prepared Thermo Scientific CloneKet PCR Cloning Kit reagent mix in a PCR tube on ice
2. Added 10μL 2X Reaction Buffer
3. Added 1μL resuspended pcaA gBlock gene fragement
4. Added
1μL pJET 1.2/blunt cloning vector (50ng/μL)
5. Added 7μL nuclease-free water
6. Added 1μL T4 DNA Ligase
7. Incubated at room temperature for 16 hours
8. Repeated for HBcAg and IS481 gBlocks
Transformation of E.coli via heat shock using ligated pJET vectors incubated for 16 hours
1. Aliquoted 33μL of electrocompetent E.coli into 3 eppendorf tubes
2. Added either 10μL of pcaA, HBcAg or IS481 ligated pJET vectors into each of 3 eppendorf tubes
3. Iced for
20 minutes,
4. 60 second heat shock in eppendorf heat block at 42°C
5. Returned tubes to ice for 2 minutes
6. Added 800μL of SOC
7. Incubated on shaker
at 220 rpm and 37°C for one hour
8. Centrifuged for 1 minute at 13000rpm and discarded 700μL of supernatant
9. Carefully resuspended the pellet and plated on LB+AMP agar
10. Spreaded
gently using plastic spreader
11. Incubated overnight (16 hours) at 37 °C
Results of Ligation and Transformation using heat shock and overnight ligation incubation:
Growth on plates.
Wednesday : 7/17/2019
Inoculation of pcaA, HBcAg and IS481 colonies from transformation
Inoculation steps:
1. 5mL LB broth was added to 3 15mL culture tubes
2. A plastic inoculation loop was used to select a colony from each plate and was swirled in the corresponding broth to dislodge the
colony
3. The
tubes were loosely capped and incubated on a shaker at 220rpm and 37°C overnight (16 hours)
Thursday : 7/18/2019
Mini-prepped inoculated pcaA, HBcAg and IS481 samples
DNA extraction:
1. Pellet 1–5 ml bacterial overnight culture by centrifugation at >8000 rpm
(6800 x g) for 3 min at room temperature (15–25°C).
2. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and
transfer to a
microcentrifuge tube.
3. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube
4–6 times until the solution becomes clear. Do not allow the lysis reaction to
proceed for more than
5 min. If using LyseBlue reagent, the solution will turn
blue.
4. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube
4–6 times. If using LyseBlue reagent, the solution will turn
colorless.
5. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top
microcentrifuge.
October 2010
Quick-StartProtocol
Sample & Assay Technologies
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, or apply
vacuum to the manifold to draw the solution through the QIAprep spin column
and switch off
the vacuum source.
7. Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB Centrifuge for 30–60 s and discard the flow-through, or apply vacuum to
the manifold to draw the solution through
the QIAprep spin column and switch
off the vacuum source.
Note: This step is only required when using endA+ strains or other bacteria
strains with high nuclease activity or carbohydrate content.
8. Wash
the QIAprep spin column by adding 0.75 ml Buffer PE.
Centrifuge for 30–60 s and discard the flow-through, or apply vacuum to
the manifold to draw the solution through the QIAprep spin column and switch
off the vacuum
source. Transfer the QIAprep spin column to the collection tube.
9. Centrifuge for 1 min to remove residual wash buffer.
10. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube.
To elute DNA,
add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of the
QIAprep spin column, let stand for 1 min, and centrifuge for 1 min
A nanodrop was used to measure the concentration of the plasmids.
A gel electropheresis was run to confirm the ligation of the gblocks into the pJET vectors.
Resuspension, ligation and transformation of ypo2088 gblock
Resuspended ypo2088 gBlock gene fragment from IDT :
1. For 500ng gBlocks, 50μL of molecular grade water was added to the tube. For 1000ng gBlocks, 100μL of molecular grade water was added to the tube to reach a final concentration of 10ng/μL
2. The
tubes were vortexed briefly then incubated at 50°C for 15 mins
3. The tubes were briefly vortexed and centrifuged for 5 seconds
4. The concentration of the gBlock resuspension was taken with
the NanoDrop
Resupension Results:
Transformation with Heat Shock and 2 hour ligation incubation
pJET Blunt End Ligation
1. Prepared Thermo Scientific CloneKet PCR Cloning Kit reagent mix in a PCR tube on ice
2. Added 10μL 2X Reaction Buffer
3. Added 1μL resuspended ypo2088 gBlock gene fragement
4. Added
1μL pJET 1.2/blunt cloning vector (50ng/μL)
5. Added 7μL nuclease-free water
6. Added 1μL T4 DNA Ligase
7. Incubated at room temperature for 16 hours
Incubate DNA at room temperature for two hours at room temperature.
Transformation of E.coli with ypo2088 via heat shock using ligated pJET vectors incubated for 16 hours
1. Added 50 μL of electrocompetent E.coli into eppendorf tube
2. Added 10μL of ypo2088 ligated pJET vectors into the eppendorf tube
3. Iced for 20 minutes, followed by a 60 second
heat shock in eppendorf heat block at 42°C
4. Returned tibes to ice for 2 minutes
5. Added 800μL of SOC
6. Incubated on shaker at 220 rpm and 37°C for one hour
7. Centrifuged
for 1 minute at 13000rpm and discarded 700μL of supernatant
8. Carefully resuspended the pellet and plated on LB+AMP agar. Less than or around 20 μL of the resuspended pellet were added to the plate.
9. Spreaded
gently using plastic spreader
10. Incubated overnight (16 hours) at 37 °C
Results of Ligation and Transformation using heat shock and overnight ligation incubation:
Formation of very few and small colonies after overnight incubation.
Friday : 7/19/2019
Transformation with Heat Shock after 24 hour ligation incubation at room temperature of remaining 10μL of ypo2088 DNA and inoculation of ypo2088 transformed E.coli colonies from 7/18 transformation
Transformation of E.coli with ypo2088 via heat shock using ligated pJET vectors incubated for 16 hours
1. Added 50 μL of electrocompetent E.coli into eppendorf tube
2. Added 10μL of ypo2088 ligated pJET vectors into the eppendorf tube
3. Iced for 20 minutes, followed by a 60 second
heat shock in eppendorf heat block at 42°C
4. Returned tibes to ice for 2 minutes
5. Added 800μL of SOC
6. Incubated on shaker at 220 rpm and 37°C for one hour
7. Centrifuged
for 1 minute at 13000rpm and discarded 700μL of supernatant
8. Carefully resuspended the pellet and plated on LB+AMP agar. Less than or around 20 μL of the resuspended pellet were added
to the plate.
9. Spreaded gently using plastic spreader
10. Incubated overnight (16 hours) at 37 °C
Inoculation of ypo2088 transformed E.coli colonies from transformation on Thurs 7/18:
1. 5mL LB broth was added to 2 15mL culture tubes
2. A plastic inoculation loop was used to select a colony from each plate and was swirled in the corresponding broth to dislodge the
colony
3. The tubes were loosely capped
and incubated on a shaker at 220rpm and 37°C overnight (16 hours)
