Daniel:Notebook/ComboLock/2017-2-2
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RCA Time Test (Started Friday Jan 27)
Phosphorothiorate RCA Primer (Started Yesterday)
- qPCR
- Make qPCR master mix according to following recipe
- 361.2 uL nfH2O
- 17.2 uL AmpF6.4
- 430 uL Kapa SYBR Fast
- Add 47 uL master mix to each well
- Add 2 uL sample and 1 uL AmpR indexed according to table
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x24
- 72C 2 min
- 16C hold
- TBE Gel
- Mix 94 uL TBE, 24 uL 6x loading dye
- Aliquot 10 uL per sample/ladder lane onto parafilm
- Add 2 uL of sample or ladder to correct drop
- Load 10 uL in to well
- Run gel for 23 minutes at 230V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
Sample | Sample | Condition | AmpR Index | Lane | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 10 uM Forward Primer | 10 uM Reverse Primer | H2O | Total Volume (uL) |
Sample 1AN | Sample | 3 hours | 20 | A8 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1BN | Sample | 3 hours | 20 | A7 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3AN | No C Probe | 3 hours | 21 | A6 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3BN | No C Probe | 3 hours | 21 | A5 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1AO | Sample | 3 hours-Epi | 22 | A4 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1BO | Sample | 3 hours-Epi | 22 | A3 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3AO | No C Probe | 3 hours-Epi | 23 | A2 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3BO | No C Probe | 3 hours-Epi | 23 | A1 | 2 | 25 | 1 | 1 | 21 | 50 |
NTC | 24 | H1 | 0 | 25 | 1 | 1 | 23 | 50 |
Results
- 20170202-qPCR-RCATimeTest-Phosphorothiorate.png
qPCR curve