Matt:LabNotes/2013-5-11
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Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-5-10
Expansion PCR
- Made 2 Master mixes (for 0 gap and 20 gap probes)
- Each MM is for 46 well (half a 96 well plate)
- 0 gap first round amplicon -> AP1V6 primers
- 20 gap first round amplicon -> AP1V4 primers
- First round amplicons needed to be diluted to 10nM so made 12ul dilutions of each
0 gap: 12ul x 10nM = 1.07ul * 112nM -> 1.071ul first round amplicon + 10.929 H20 20 gap: 12ul * 10nM = 0.98ul * 123nM -> 0.976ul first round amplicon + 11.024 H2O
Components | 1x rxn (ul) | 50x rxn (ul) |
First round amplicon (10nM) | 0.2 | 10 |
2x Kapa SYBR MM | 50 | 2500 |
Forward primer (100uM) | 0.4 | 20 |
Reverse primer (100uM) | 0.4 | 20 |
H2O | 49 | 2450 |
Total | 100 | 5000 |