Daniel:Notebook/ComboLock/2016-9-26
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PCR Testing (Started Thursday)
Continuing the tests on qPCR for the positive control amplicon using primer concentrations. This is following some of the conclusions from Friday.
Protocol
- qPCR
- Follow reaction map below
- Make master mixes according to following table
- Add 48 uL correct master mix to each well
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x26
- 72C 2 min
- 16C hold
Sample | Molecule Count | Lane | AmpR Index | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 10 uM AmpF 6.4 | 10 uM AmpR6.3-IndXX | H2O | Total Volume (uL) | Master Mix |
Sample 1A | 1.00E+09 | A1 | 36 | 2 | 25 | 5 | 5 | 13 | 50 | MM1 |
Sample 1B | 1.00E+09 | A2 | 36 | 2 | 25 | 5 | 5 | 13 | 50 | MM1 |
Sample 1C | 1.00E+09 | A3 | 36 | 2 | 25 | 5 | 5 | 13 | 50 | MM1 |
NTC | 0 | A4 | 36 | 0 | 25 | 5 | 5 | 13 | 50 | NTC |
Sample 1A | 1.00E+09 | A5 | 36 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 1B | 1.00E+09 | A6 | 36 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 1C | 1.00E+09 | A7 | 36 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
NTC | 0 | A8 | 36 | 0 | 25 | 1 | 1 | 21 | 50 | NTC |
Sample 1A | 1.00E+09 | H1 | 36 | 2 | 25 | 0.5 | 0.5 | 22 | 50 | MM3 |
Sample 1B | 1.00E+09 | H2 | 36 | 2 | 25 | 0.5 | 0.5 | 22 | 50 | MM3 |
Sample 1C | 1.00E+09 | H3 | 36 | 2 | 25 | 0.5 | 0.5 | 22 | 50 | MM3 |
NTC | 0 | H4 | 36 | 0 | 25 | 0.5 | 0.5 | 22 | 50 | NTC |
Sample 1A | 1.00E+09 | H5 | 37 | 2 | 25 | 2* | 2* | 19 | 50 | MM4 |
Sample 1B | 1.00E+09 | H6 | 37 | 2 | 25 | 2* | 2* | 19 | 50 | MM4 |
Sample 1C | 1.00E+09 | H7 | 37 | 2 | 25 | 2* | 2* | 19 | 50 | MM4 |
NTC | 0 | H8 | 37 | 0 | 25 | 2* | 2* | 19 | 50 | NTC |
Sample 1A | 1.00E+09 | C1 | 37 | 2 | 25 | 0.2* | 0.2* | 22.6 | 50 | MM5 |
Sample 1B | 1.00E+09 | C2 | 37 | 2 | 25 | 0.2* | 0.2* | 22.6 | 50 | MM5 |
Sample 1C | 1.00E+09 | C3 | 37 | 2 | 25 | 0.2* | 0.2* | 22.6 | 50 | MM5 |
NTC | 0 | C4 | 37 | 0 | 25 | 0.2* | 0.2* | 22.6 | 50 | NTC |
Reagent | Master Mix | ||||
MM1 | MM2 | MM3 | MM4 | MM5 | |
PrimerF (10 uM) | 15.5 | 3.1 | 1.55 | 6.2* | 0.62* |
PrimerR (10 uM) | 15.5 | 3.1 | 1.55 | 6.2* | 0.62* |
KAPA Master Mix | 77.5 | 77.5 | 77.5 | 77.5 | 77.5 |
nfH2O | 40.3 | 65.1 | 68.2 | 58.9 | 70.06 |