Daniel:Notebook/ComboLock/2016-10-27
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Cell Test 3 (Started Friday 10/21)
Latch PCR
Just testing the PCR of Latch0001, which didn't show up at all in the gels from Tuesday. I'm just going to test on the final product, but I'll try to make sure that we get the cells from the bottom of the tubes.
- qPCR
- Prepare the following master mix
- 128.1 uL nfH2O
- 6.1 uL primer4
- 6.1 uL primer6RC
- 152.5 uL Kapa SYBR Fast master mix
- Add 48 uL master mix to each well
- Add 2 uL sample to correct well by following sample 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 200 uL TBE, 50 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 22 minutes at 250V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
Sample | Condition | Lane | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 10 uM p6RC (fwd) Primer | 10 uM p4 (rev) Primer | H2O | Total Volume (uL) |
Sample 1 | Exp. Rep 1 | A1 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2 | Exp. Rep 2 | B1 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3 | No C Probes | C1 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4 | No Cells | D1 | 2 | 25 | 1 | 1 | 21 | 50 |
NTC | qPCR primers only | E1 | 0 | 25 | 1 | 1 | 21 | 50 |
Positve Control | Direct Latch0001 (10 uM) | F1 | 2 | 25 | 1 | 1 | 21 | 50 |