Daniel:Notebook/ComboLock/2016-12-1: Difference between revisions
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>Djacobse (Created page with "=Extended Latch Test (Started Nov 29)= Back to Calendar ===Sample Matrix=== <li>Rolling Circle Ampl...") |
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Revision as of 18:27, 1 December 2016
Extended Latch Test (Started Nov 29)
Sample Matrix
- Rolling Circle Amplification
- Prepare 9X master mix according to table below
- Add 15 uL master mix to each tube
- Add 5 uL sample to correct reaction tubes
- Incubate at 37C for 3 hours
- Heat kill by incubating at 65C for 10 minutes
- qPCR
- Make master mix according to following recipe
- 359.1 uL nfH2O
- 427.5 uL SYBR Fast
- 17.1 uL 10 uM AmpF
- Add 47 uL master mix to each well
- Add 2 uL sample and 1 uL appropriate Reverse Primer (1 uM) 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 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
Reagent | Stock Concentration | Final Conc/Amt | uL Added in 20 uL Rxn | Master Mix (9X) |
Template | NA | NA | 5 | 45 |
RCA Primer (RCA-CL-LinkerLRC-v01) | 10 uM | 2 uM | 4 | 36 |
dNTP | 1 mM | 50 uM | 1 | 9 |
10X Buffer | 10X | 1X | 2 | 18 |
Phi29 (NEB) | 10 U/uL | 10 U | 1 | 9 |
nfH2O | NA | NA | 7 | 63 |
Total | 20 | 180 |
Sample | Condition | RCA | Lane | AmpR Index | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 10 uM Forward Primer | 10 uM Reverse Primer | H2O | Total Volume (uL) |
Sample 1A | Sample | Y | A1 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1B | Sample | Y | A2 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2A | No Template | Y | A3 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2B | No Template | Y | A4 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3A | No Phosphate | Y | A5 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3B | No Phosphate | Y | A6 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4A | No C Probe | Y | A7 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4B | No C Probe | Y | A8 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1A | Sample | N | A1 | 24 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1B | Sample | N | A2 | 24 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2A | No Template | N | A3 | 25 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2B | No Template | N | A4 | 25 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3A | No Phosphate | N | A5 | 26 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3B | No Phosphate | N | A6 | 26 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4A | No C Probe | N | A7 | 27 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4B | No C Probe | N | A8 | 27 | 2 | 25 | 1 | 1 | 21 | 50 |
NTC | qPCR Primers Only | NA | H1 | 28 | 2 | 25 | 1 | 1 | 23 | 50 |