Daniel:Notebook/ComboLock/2017-3-29
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RCA Test (Started March 24)
Bglii Digestion
Testing bglii digestion of RCA product. No qPCR to start with (RCA should be enough). For recall, here is the sample matrix again and the RCA conditions used.
Sample | Master Mix | Enzyme | Incubation Time (hrs) |
X (A-F) | X | NEB | 3 |
Y (A-F) | Y | Thermo | 3 |
Z (A-F) | Z | Epi (1:10) | 3 |
P (A-F) | X | NEB | 6 |
Q (A-F) | X | NEB | 12 |
- Bglii digestion
- Make the following reactions; Do not add Bglii yet
- Use the following thermocycler program
- 5 min 95C
- Ramp to 50C at 0.2C/s
- 10 min 50C
- 2 hr 37C; When this cycle starts add 1 uL Bglii
- Heat kill with 65C for 20 min
Reagent | Single Rxn Vol | Master Mix (30.2X) |
RCA Rxn | 2 | 0 |
Bglii Cutter Primer + (100 uM) | 2 | 60.4 |
10X Buffer 3.1 | 2 | 60.4 |
Bglii | 1 | 0 |
nfH2O | 13 | 392.6 |
Total | 20 | 513.4 |