Daniel:Notebook/ComboLock/2017-3-21
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Circularization Comparison (T4 Poly/Ligase; Started Wednesday)
Bgl Digestion
Because I CAN. Also, will determine if there is an improvement in signal post-digestion.
Sample Matrix
Sample Origin | Condition | Sample Numbers |
F | T4 Ligase | 1,4 |
G | Phusion/Amp (Unpurified) | 1,4 |
H | Phusion/AmpHS (Unpurified) | 1,4 |
- Bglii Digestion
- Use samples 1 (Experiment) and 4 (No phosphate) for Bglii digest
- Make the following 4.2X master mix
- Aliquot 14 uL master mix into new tubes
- Add 5 uL appropriate sample to each tube
- Use the following thermocycler program
- 5 min 95C
- 10 min 50C
- 1 hr 37C; When this cycle starts add 1 uL Bglii
- Heat kill with 65C for 20 min
Reagent | Single Rxn Vol | 14.2X MM uL |
RCA Reaction Product | 5 | 0 |
Bglii Cutter Primer (10 uM) | 5 | 71 |
10X Buffer 3.1 | 2 | 28.4 |
Bglii | 1 | 14.2 |
nfH2O | 7 | 99.4 |
Total | 20 | 198.8 |