Daniel:Notebook/ComboLock/2017-4-12
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RCA Double Deuce (Started Yesterday)[edit]
Protocol[edit]
- Bglii digestion
- Make the following reactions; Do not add Bglii yet
- Aliquot 12 uL master mix into new tube
- Add 7 uL appropriate sample
- Mix with vortexer, spin down, and add 1 uL Bglii
- Incubate 2 hr 37C
- Heat kill with 65C for 20 min
- TBE Gel
- Mix 120 uL 10X TBE and 40 uL 6X dye
- Aliquot 8 uL TBE/dye mix onto parafilm per sample
- Add 4 uL sample to sample lanes
- Mix 8 uL 1X TBE, 2 uL 6X dye, and 1.5 uL 25bp ladder for ladder sample
- Add 10 uL from aliquots into appropriate gel lanes
- Run gel for 25 minutes at 250V
- Stain with 2 uL SYBR gold for 3 min
- Rinse and image in gel doc
Reagent | Single Rxn Vol | Master Mix (13.2X) |
RCA Rxn | 7 | 0 |
10X Buffer 3.1 | 2 | 26.4 |
Bglii | 1 | 0 |
nfH2O | 10 | 132 |
Total | 20 | 158.4 |
- 2017-04-12-RCADD-NEB.png
Samples amplified with NEB Phi29
- 2017-04-12-RCADD-Thermo.png
Samples amplified with Thermo Phi29