Daniel:Notebook/HiResChrPaint/2014-8-18
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Padlock Probes (Started 8/13/2014)
qPCR
I need more padlock probes, so I'm going to repeat qPCR with 24x samples.
Reaction Table
Component | 1X Reaction | 12.2X Scaled |
Padlock Probes | 0.2 | 2.44 |
100 uM pAP1V41U | 0.4 | 4.88 |
100 uM pAP2V4 | 0.4 | 4.88 |
KAPA SYBR Fast Master Mix (2X) | 50 | 610 |
H2O | 49 | 597.8 |
Total | 100 | 1220 |
- Prepare master mix according to table above
- Add 100 uL reaction to PCR tubes (5 total)
- qPCR Protocol
- 95C 30 sec
- 95C 10 sec
- 55C 20 sec
- 72C 30 sec
- goto (3.2) 17 times
- 72C 2 min
- Hold at 16C forever
- Ethanol Precipitation
qPCR Results
USER/DpnII Digestion
Reaction Table
Reagent | uL |
amplicon | 35 |
USER | 5 |
nf H2O | 40 |
Total | 80 |
- Set up reaction according to table in a 0.2 mL PCR tube
- Incubate for 2 hours at 37C
- Add 15 uL according to DpnII Primer table (below)
DpnII Primer Table
Reagent | uL |
10X DpnII Buffer | 10 |
100 uM RE-DpnII-V4 guide oligo | 5 |
Total | 15 |
- Incubate at 94C for 2 minutes
- Cool to 37C and incubate for 3 minutes
- Add 5 uL DpnII and incubate at 37C for 2 hours
- Heat inactivate DpnII at 65C for 20 minutes