Daniel:Notebook/ComboLock/2017-1-28
Jump to navigation
Jump to search
RCA Timing Test (v3 Oligos)
Protocol
- 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 in following section
- 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 280 uL TBE, 70 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
qPCR Table
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 1A | Sample | PreRCA | 10 | A1 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1B | Sample | PreRCA | 10 | A2 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2A | No Template | PreRCA | 11 | A3 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2B | No Template | PreRCA | 11 | A4 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3A | No C Probe | PreRCA | 12 | A5 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3B | No C Probe | PreRCA | 12 | A6 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4A | No PO4 | PreRCA | 13 | A7 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4B | No PO4 | PreRCA | 13 | A8 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1AX | Sample | 3 hours | 14 | H1 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1BX | Sample | 3 hours | 14 | H2 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2AX | No Template | 3 hours | 15 | H3 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2BX | No Template | 3 hours | 15 | H4 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1AY | Sample | 6 hours | 16 | H5 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1BY | Sample | 6 hours | 16 | H6 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3AY | No Template | 6 hours | 17 | H7 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3BY | No Template | 6 hours | 17 | H8 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1AZ | Sample | 12 hours | 18 | C1 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1BZ | Sample | 12 hours | 18 | C2 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4AZ | No Template | 12 hours | 19 | C3 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4BZ | No Template | 12 hours | 19 | C4 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1AZ | Sample | 3 hours-Epicentre | 20 | C5 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1BZ | Sample | 3 hours-Epicentre | 20 | C6 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4AZ | No Template | 3 hours-Epicentre | 21 | C7 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4BZ | No Template | 3 hours-Epicentre | 21 | C8 | 2 | 25 | 1 | 1 | 21 | 50 |
NTC | None | 22 | F1 | 0 | 25 | 1 | 1 | 23 | 50 |
Results
- 20170128-qPCR-RCATimeTest-All.png
qPCR Curve-All Samples
- 20170128-qPCR-RCATimeTest-SamplesOnly.png
qPCR Curve-Sample Lanes Only
- 2017-01-28-RCATimeTest-PreRCA.png
Gel image-Pre RCA
- 2017-01-28-RCATimeTest-3and6hr.png
Gel image-Post RCA #1
- 2017-01-28-RCATimeTest-12hrandEpi.png
Gel image-Post RCA #2