Daniel:Notebook/ComboLock/2017-4-29
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Production Run (Started Yesterday)
Protocol-Part 4
- Exonuclease Digestion
- Mix 25 uL Exo I (20U/uL) and 5 uL ExoIII (100U/uL)
- Add 2 uL to each sample, mix by swirling pipette tip
- Incubate for 1.5 hours at 37C
- Heat kill by incubating for 5 min at 95C
- Rolling Circle Amplification
- Prepare master mix according to table below
- Add 15 uL master mix to each tube
- Samples "A-C" get + master mixes; samples "D-F" get - master mixes
- Add 1 uM RCA primer and to new reaction tubes according to following rules
- "A,D" samples get 2 uL each LLRC & primer12RC
- "B,E" samples get 2 uL each LLRC*** & primer12RC
- "C,F" samples get 4 uL LLRC***
- Heat reactions up to 95C for 5 min
- Cool to 55C and incubate for 15 minutes
- Cool to 30C and add enzyme
- Incubate for 3 hours at 30C
- Incubate for 65 minutes at 20C to denature
Reagent | Single Rxn (uL) | Master Mix X (10.2X) | Master Mix Y (6.2X) |
Enzyme | NA | NEB | Thermo |
Sample | 4 | 0 | 0 |
RCA Primer (1 uM each) | 2 | 20.4 | 12.4 |
dNTP (1 mM) | 5 | 51 | 31 |
10X Buffer | 2 | 20.4 | 12.4 |
Phi29 | 1 | 10.2 | 6.2 |
BSA (10 mg/mL) | 0.4 | 4.08 | 0 |
nfH2O | 5.6 | 57.12 | 37.2 |
Total | 20 | 163.2 | 99.2 |
- Make qPCR master mixes according to following recipe