Daniel:Notebook/ComboLock/2017-1-28
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RCA Timing Test (v3 Oligos)[edit]
Protocol[edit]
- 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[edit]
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[edit]
- 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
CT Table[edit]
Sample No | Condition | CT | PreRCA Sample | Fold Diff Over PreRCA | Conc. Difference |
1AZ | 12 hours | 11.52 | 1A | 7.3 | 153.3 |
4BZ | 12 hours | 11.52 | 4B | 6.3 | 78.2 |
1BZ | 12 hours | 11.93 | 1B | 5.9 | 57.7 |
4AZ | 12 hours | 12.07 | 4A | 5.2 | 36.5 |
1BY | 6 hours | 12.38 | 1B | 5.4 | 42.2 |
1AY | 6 hours | 13.26 | 1A | 5.5 | 45.9 |
3BY | 6 hours | 14.35 | 3B | 3.5 | 11.3 |
3AY | 6 hours | 14.59 | 3A | 3.2 | 9.1 |
2BX | 3 hours | 15.16 | 2B | 2.3 | 4.8 |
1BQ | 3 hours-Epicentre | 15.17 | 1B | 2.6 | 6.1 |
2AX | 3 hours | 15.3 | 2A | 2.1 | 4.3 |
1AQ | 3 hours-Epicentre | 15.31 | 1A | 3.5 | 11.1 |
1AX | 3 hours | 15.42 | 1A | 3.4 | 10.3 |
1BX | 3 hours | 15.46 | 1B | 2.3 | 5.0 |
4BQ | 3 hours-Epicentre | 16.37 | 4B | 1.4 | 2.7 |
4AQ | 3 hours-Epicentre | 17.07 | 4A | 0.2 | 1.1 |
4A | PreRCA | 17.26 | 4A | 0.0 | 1.0 |
2A | PreRCA | 17.39 | 2A | 0.0 | 1.0 |
2B | PreRCA | 17.43 | 2B | 0.0 | 1.0 |
3A | PreRCA | 17.77 | 3A | 0.0 | 1.0 |
1B | PreRCA | 17.78 | 1B | 0.0 | 1.0 |
4B | PreRCA | 17.81 | 4B | 0.0 | 1.0 |
3B | PreRCA | 17.85 | 3B | 0.0 | 1.0 |
1A | PreRCA | 18.78 | 1A | 0.0 | 1.0 |