Matt:LabNotes/2013-5-11
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Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-5-10
Production PCR[edit]
- 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 |
98C 30sec -> (98C 10sec -> 55C 20sec -> 72C 30sec) x 16 -> 72C 2min -> 15C hold
- Stopped after 15 runs
File:051113 ProductionPCR Agi26k.jpg
- Lower fluorescence samples are due to wells with less than 100ul (even with making 4 extra mastermix samples in previous step, wasn't able to pipette a full 100ul in the last 1-3 wells)
EtOH Precipitation[edit]
- 6 15-ml tubes (with 8 wells of PCR product each) for 0gap and 6 tubes for 20gap
- 800ul PCR product
- 2000ul 100% EtOH
- 2.7ul GlycoBlue
- 80ul 3M NaOAc pH 5.2-5.5
- Vortexed and put in -80C for 1hr
- Centrifuged at 3000rpm at 4C for 30 min
- Discarded supernatant and added 800ul of cold 80% EtOH
- Transferred DNA pellet (with 1ml pipette tip) to 12 1.5mL tubes
- Centrifuged 1.5ml tubes at 15,000rpm for 5 min on table-top centrifuge
- Discarded supernatant and air-dried for 5 min in hood
- Resuspended DNA with 100ul H2O
Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-5-12