Daniel:Notebook/ComboLock/2016-11-30
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Extended Latch Test (Started [[Daniel:Notebook/ComboLock/2016-11-29])
Sample Matrix
Sample | Condition |
Sample 1 (AB) | Normal |
Sample 2 (AB) | No Template |
Sample 3 (AB) | No C Probe |
Sample 4 (AB) | No Kinase |
Protocol
- Exonuclease Digestion
- Combine 15 uL of Exonuclease I (20U/uL) and 3 uL Exonuclease III (100U/uL)
- Add 2 uL of mix to each reaction
- Incubate for 30 minutes to 2 hours at 37C
- Heat kill by incubating for 5 minutes at 94C
- Rolling Circle Amplification
- Prepare 9X master mix according to table below
- Add 15 uL master mix to each tube
- Add 5 uL sample to correct reaction tubes
- Incubate at 37C for 3 hours
- Heat kill by incubating at 65C for 10 minutes
Reagent | Stock Concentration | Final Conc/Amt | uL Added in 20 uL Rxn | Master Mix (9X) |
Template | NA | NA | 5 | 45 |
RCA Primer | 10 uM | 2 uM | 4 | 36 |
dNTP | 1 mM | 50 uM | 1 | 9 |
10X Buffer | 10X | 1X | 2 | 18 |
Phi29 (NEB) | 10 U/uL | 10 U | 1 | 9 |
nfH2O | NA | NA | 7 | 63 |
Total | 20 | 180 |