Daniel:Notebook/HiResChrPaint/2014-8-13
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Padlock Probe Production[edit]
Rui has a set of padlock probes that target chromosome 21 in bulk. Hopefully, by using these, we will be able to see strong signal. I'll label them using the ULYSIS system, which should non-specifically label the Guanines. For production, I will be following (and occasionally modifying) Noi's instructions on padlock probe preparation: Custom Array Probe Prep Protocol
Test qPCR[edit]
To test the qPCR step (make sure the primers I'm using are the right ones, as well as just do a small scale PCR before I use 20+ reactions worth. One qPCR should also give me an expectation for number of cycles and expected yield.
Reaction Matrix
Component | 1X Reaction | 5.1X Scaled |
Padlock Probes | 0.2 | 1.02 |
100 uM pAP1V41U | 0.4 | 2.04 |
100 uM pAP2V4 | 0.4 | 2.04 |
KAPA SYBR Fast Master Mix (2X) | 50 | 255 |
H2O | 49 | 249.9 |
Total | 100 | 510 |
- The 5X scaling is to give myself n=5 to estimate yields with more power (and because 0.2 uL is impossible to add). The other 0.1 is for additional volume, so 100 uL may be added to each well.
- Fill 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?
Results[edit]
qPCR Results
Successful qPCR Amplification, stopped cycle 13
Nanodrop Results
Sample | ng/uL | ug in 40 uL | Total |
PPRL-A | 71.3 | 2.9 | |
PPRL-B | 71.3 | 2.9 | 5.7 |