Daniel:Notebook/ComboLock/2017-2-3
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Version 2 Oligos Round 2 (Started yesterday)
Protocol-Part 4-RCA
- Exonuclease Digestion
- Mix 15 uL Exo I (20U/uL) and 3 uL ExoIII (100U/uL)
- Add 2 uL to each sample, mix by swirling pipette tip
- Incubate for 1 hour at 37C
- Heat kill by incubating for 5 min at 95C
- Rolling Circle Amplification
- Prepare 8.2X Master mix according to table below
- Add 14 uL master mix to each tube
- Add sample and 1 uL appropriate enzyme to new reaction tubes
- Incubate at 37C for 3 hours
- Incubate at 65C for 10 minutes;Hold at 10C until next step
Reagent | uL Added | Master Mix (8.2X) | Master Mix (4.2X) |
Template | 5 | NA | NA |
RCA Primer (10 uM) | 2.5 | 20.5 | 10.5 |
dNTP (1 mM) | 5 | 41 | 21 |
10X Buffer | 2 | 16.4 | 8.4 |
Phi29 | 1 | 0 | 0 |
BSA (10 mg/mL) | 0.4 | 3.28 | 0 |
nfH2O | 4.1 | 33.62 | 18.9 |
Total | 20 | 114.8 | 58.8 |
Sample | 1X (AB) | 2X (AB) | 3X (AB) | 4X (AB) | 1Y (AB) | 4Y (AB) |
Phi29 Source | NEB | NEB | NEB | NEB | Epicentre | Epicentre |
Protocol Part 5-qPCR and Gel
- qPCR
- Make 9.2X qPCR master mix according to following recipe
- 193.2 uL nfH2O
- 230 uL SYBR Fast
- 9.2 uL 10 uM AmpF
- Add 47 uL master mix to each well
- Add 2 uL sample and 1 uL appropriate Reverse Primer (1 uM) according to table in the following section
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x24
- 72C 2 min
- 16C hold
- TBE Gel
- Mix 80 uL TBE, 20 uL 6x loading dye
- Aliquot 10 uL per sample/ladder lane onto parafilm
- Add 2 uL of sample or ladder to correct drop
- Load 10 uL in to well
- Run gel for 22 minutes at 250V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc