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 4d |
" |
329 |
~180, 149
|
| mCherry |
blank |
" |
329 |
no cutting (PCR already done)
|
| luciferase gRNA20 |
g20 +dox |
gRNA 021 US P F / gRNA 027 ES P R |
1295 |
~165, 1130
|
| luciferase gRNA20 |
g20 -dox |
" |
1295 |
~165, 1130
|
| luciferase gRNA29 |
g29 +dox |
" |
1295 |
~395, 900
|
| luciferase gRNA29 |
g29 -dox |
" |
1295 |
~395, 900
|
| luciferase |
mock +dox |
" |
1295 |
no cutting
|
| luciferase |
mock -dox |
" |
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 1d |
1.562 |
1.81 |
78.1
|
| CRISPR 2d |
0.273 |
1.71 |
14.0
|
| CRISPR 4d |
1.77 |
1.588 |
79.3
|
| g20 +dox |
1.408 |
1.79 |
70.4
|
| g20 -dox |
1.057 |
1.78 |
52.9
|
| g29 +dox |
0.987 |
1.77 |
49.4
|
| g29 -dox |
0.856 |
1.82 |
42.8
|
| +dox |
1.300 |
1.77 |
65.0
|
| -dox |
1.169 |
1.8 |
58.5
|
| Blank 1+3 |
--- |
--- |
~22.0
|
- Combined Blank 1 and Blank 3, ~22 ng/μL in 100 uL volume
Mismatch annealing
- 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 20 μL
- 200 ng CRISPR "1"
- 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 ∞
|
Project name
