Daniel:Notebook/ComboLock/2017-1-14
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Version 3 Oligos (Started Yesterday)[edit]
Protocol-Part3[edit]
- Exonuclease Digestion
- Mix 15 uL Exo I (20U/uL) and 3 uL ExoIII (100U/uL)
- Add 2 uL to each sample, mix by swirling pipette tip
- Incubate for 1 hour at 37C
- Heat kill by incubating for 5 min at 95C
- Rolling Circle Amplification
- Prepare 7X Master mix according to table below
- Add 15 uL master mix to each tube
- Add sample to new reaction tubes according to following table
- Incubate at 37C for 3 hours
- Incubate at 65C for 10 minutes;Hold at 10C until next step
- qPCR
- Make 9.2X qPCR master mix according to following recipe
- 193.2 uL nfH2O
- 230 uL SYBR Fast
- 9.2 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 80 uL TBE, 20 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 | uL Added | Master Mix (8.5X) |
Template | 5 | NA |
RCA Primer (10 uM) | 2.5 | 21.25 |
dNTP (1 mM) | 0.8 | 6.8 |
10X Buffer | 2 | 17 |
Phi29 | 1 | 8.5 |
nfH2O | 8.7 | 73.95 |
Total | 20 | 127.5 |
Protocol-Part4[edit]
Sample | Condition | 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 | A1 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1B | Sample | A2 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2A | No Template | A3 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2B | No Template | A4 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3A | No C Probe | A5 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3B | No C Probe | A6 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4A | No Lock Phosphate | A7 | 24 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4B | No Lock Phosphate | A8 | 24 | 2 | 25 | 1 | 1 | 21 | 50 |
NTC | qPCR Primers Only | H1 | 25 | 2 | 25 | 1 | 1 | 23 | 50 |
Results[edit]
- 20170114-qPCR-v3Oligos.png
qPCR Curve
- 2017-01-14-v3Oligos.png
Gel Image