Daniel:Notebook/ComboLock/2017-2-3
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Version 2 Oligos Round 2 (Started yesterday)
Protocol-Part 4-RCA
- 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 14 uL master mix to each tube
- Add sample to new reaction tubes
- Incubate at 37C for 3 hours
- Incubate at 65C for 10 minutes;Hold at 10C until next step
Reagent | uL Added | Master Mix (8.2X) |
Template | 5 | NA |
RCA Primer (10 uM) | 2.5 | 20.5 |
dNTP (1 mM) | 5 | 41 |
10X Buffer | 2 | 16.4 |
Phi29 | 1 | 0 |
BSA (10 mg/mL) | 0.4 | 3.28 |
nfH2O | 4.1 | 33.62 |
Total | 20 | 131.2 |
Protocol Part 5-qPCR and Gel
- 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 in the following section