08/05/14
- SURVEYOR assay - for mCherry CRISPR
- qPCR melt curve optimization - mCherry CRISPR 1
SURVEYOR assay
Assistance from Week 2 Team 1
PCR primers [Rene, please add this info]
PCR templates
- CRISPR "1"
- CRISPR "2"
- CRISPR "4"
- "Blank" (3x)
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
|
CRISPR "4" |
--- |
1.85 |
9.8
|
Blank 1 |
--- |
1.80 |
22.9
|
Blank 2 |
--- |
1.54 |
5.0
|
Blank 3 |
--- |
1.85 |
21.5
|
- 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)
- Temperature program
- 95ºC/ 10 min
- 95ºC -- (‐2.0 ºC/s) --> 85ºC
- 85ºC/ 1 min
- 85ºC -- (‐0.3 ºC/s) --> 75ºC
- 75ºC/ 1 min
75 ºC to 65 ºC (‐0.3 ºC/s)
65 ºC 1 min
65 ºC to 55 ºC (‐0.3 ºC/s)
55 ºC 1 min
55 ºC to 45 ºC (‐0.3 ºC/s)
45 ºC 1 min
45 ºC to 35 ºC (‐0.3 ºC/s)
35 ºC 1 min
35 ºC to 25 ºC (‐0.3 ºC/s)
25 ºC 1 min
4 ºC Hold ∞
qPCR melt curve optimization - mCherry CRISPR 1
Assistance from Kumaran Lleng
Optimization parameters
- Borun et al reported cleaner peak profiles after a 30-cycle amplification (vs. 40 cycles)
- We will use 30 cycles instead of 45
- Borun et al used at LEAST 50 ng genomic DNA in their reactions (final amount per PCR ambiguous)
- We will test 20 ng, 50 ng, and 70 ng (70 is our upper limit because of the concentration of the Blank DNA sample)
Master reaction list
Rxn #
|
Template
|
Target (primers)
|
Method
|
1 |
1 20 ng |
q2F_q3R |
CNV melt
|
2 |
1 20 ng |
GAPDH B2 |
"
|
3 |
1 50 ng |
q2F_q3R |
"
|
4 |
1 50 ng |
GAPDH B2 |
"
|
5 |
1 70 ng |
P3F_q3R |
"
|
6 |
1 70 ng |
GAPDH B2 |
"
|
7 |
Blank 20 ng |
q2F_q3R |
"
|
8 |
Blank 20 ng |
GAPDH B2 |
"
|
9 |
Blank 50 ng |
q2F_q3R |
"
|
10 |
Blank 50 ng |
GAPDH B2 |
"
|
11 |
Blank 70 ng |
q2F_q3R |
"
|
12 |
Blank 70 ng |
GAPDH B2 |
"
|
Notes:
- Opaque white plates
- Final reaction volumes = 15 μL
- Each reaction had 3.0 μL of 750 nM F/R primers, 20 ng - 70 ng template DNA, 1x Roche SYBR
- Batch (master) mixes...
- Primers: Primers plus SYBR, 10.5 μL mix per relevant well, one mix per unique primer pair
- Templates: DNA plus water, 4.5 μL mix per relevant well, one mix per unique template
- Final triplicates for the 96-well plate were first made as a single batch, then aliquoted from the batch tube into the 96-well plate
- nTc for q2F_q3R and GAPDH confirmed no background signal under identical conditions on 08/04/14
Run PCR - Bio-Rad CFX96
- 95°C, 3 min
- 30x [95°C, 10 sec / 57°C, 10 sec / 72°C, 10 sec (measure)]
- 72°C, 30 sec
- Melt: 70°C -- +0.1°C/ 5 sec (measure) --> 95°C
|