Daniel:Notebook/ComboLock/2017-7-18
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Multiplex Experiment (Started Thursday July 13)[edit]
Given yesterday's results, I'm going to repeat the 6 hour RCA and also do a prep heat.
Protocol[edit]
- Rolling Circle Amplification
- Vortext samples and resuspend with pipette
- Heat for 5 minutes at 95C
- During this time, prepare master mix according to table below
- Add 19 uL master mix to each tube
- Heat reactions up to 95C for 5 min
- Cool to 55C and incubate for 15 minutes
- Cool to 30C and add 1 uL enzyme
- Incubate for 6 hours at 30C
- Incubate for 65 minutes at 20C to denature
Reagent | Single Rxn (uL) | Master Mix X (6.2X) |
Sample | 4 | 24.8 |
p12RC*** (10 uM) | 0.2 | 1.24 |
RCA Primer LLRC*** (10 uM) | 0.2 | 1.24 |
dNTP (10 mM) | 0.5 | 3.1 |
10X Buffer | 2 | 12.4 |
Phi29 | 1 | 0 |
nfH2O | 12.1 | 75.02 |
Total | 20 | 117.8 |
Continued tomorrow