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08/07/2014
- CRISPR on chromatin: transfections of g21, g22, g27 into Gal4EED/luc cells
- Luc-mCh swap: building the donor sequence
- SURVEYOR assay - C-on-C and mCherry
CRISPR on chromatin: transfections
- gRNA21/Cas9, dox+
- gRNA22/Cas9, dox+
- gRNA27/Cas9, dox+
- gRNA21/Cas9, dox-
- gRNA22/Cas9, dox-
- gRNA27/Cas9, dox-
- On 8/05/14, cells were grown in 4 mL P/S-free medium to ~80% confluence in a 6-well plate, 3 with 1 μg/mL dox, 3 without dox
Luc-mCh swap: building the donor sequence
- Designed nested PCR primers to gradually build homology arms onto mCherry. The resulting donor should...
- Place mCherry in-frame upstream of the luciferase gene
- Add an ATG start codon onto mCherry (we had to use KAH154 as a template, where mCherry has no start)
- Add a double stop (TAATAG) onto the end of mCherry
- Add 130 bp of homology with TK-luciferase to each end
SURVEYOR assay
Assistance from Week 2 Team 2
Planning
| Target |
Genomic DNA (template) |
Primers |
Amplicon (bp) |
Expected S-Cut fragments
|
| mCherry gRNA1 |
CRISPR 1d |
qPCR 1F / P3R |
329 |
~229, 100
|
| mCherry gRNA2 |
CRISPR 2d |
" |
329 |
~52, 277
|
| mCherry gRNA4 |
CRISPR 2d |
" |
329 |
~180, 149
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| mCherry |
blank |
" |
329 |
no cutting (PCR already done)
|
| luciferase gRNA20 |
g20 |
gRNA 021 US P F / gRNA 027 ES P R |
1295 |
~165, 1130
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| luciferase gRNA29 |
g29 |
" |
1295 |
~395, 900
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| luciferase |
mock |
" |
1295 |
no cutting
|
PCR reaction
- Reactions: 20 ng template DNA, 1x HF Phusion buffer, 0.2 mM dNTPs, 2.5 μL of 10 μM for each primer, 0.5 μL Phusion polymerase; in 50 μL final volume
- Program
- 98°C/ 30 sec
- 30x [98°C/ 10 sec, 58°C/ 15 sec, 72°C/ 15 sec]
- 72°C/ 10 min
- 4°C hold
PCR product clean-up
- Used QIAquick PCR Purification kit
- Elute with 50 μL H2O
| Sample |
OD 260 |
260/280 |
ng/uL
|
| CRISPR "1" |
--- |
1.82 |
40.3
|
| CRISPR "2" |
--- |
1.62 |
4.7
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| CRISPR "4" |
--- |
1.85 |
9.8
|
| Blank 1 |
--- |
1.80 |
22.9
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| Blank 2 |
--- |
1.54 |
5.0
|
| Blank 3 |
--- |
1.85 |
21.5
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- Combined Blank 1 and Blank 3, ~22 ng/μL in 100 uL volume
Mismatch annealing
- Testing 2 methods
- IDT approach - mix the mutant PCR sample with a wild type reference sample to ensure heteroduplex formation
- Zhang Lab approach - do not mix with a wild type reference, since ~50% or more of the amplicons in the mutated cell sample will be wild type anyway
- 200 ng clean PCR, 1x HF Phusion buffer, in final volume of 15 μL
- 100 ng CRISPR "1" + 100 ng Blank (IDT approach)
- 200 ng CRISPR "1" (Zhang approach)
- 200 ng Blank (wild type control)
- Thermal cycler program
- 95ºC/ 10 min
- 95ºC -- (‐2.0 ºC/s) --> 85ºC
- 85ºC/ 1 min
- 85ºC -- (‐0.3 ºC/s) --> 75ºC
- 75ºC/ 1 min
- 75ºC -- (‐0.3 ºC/s) --> 65ºC
- 65ºC/ 1 min
- 65ºC -- (‐0.3 ºC/s) --> 55ºC
- 55ºC/ 1 min
- 55ºC -- (‐0.3 ºC/s) --> 45ºC
- 45ºC/ 1 min
- 45ºC -- (‐0.3 ºC/s) --> 35ºC
- 35ºC/ 1 min
- 35ºC -- (‐0.3 ºC/s) --> 25 ºC
- 25ºC/ 1 min
- 4 ºC/ hold ∞
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