Daniel:Notebook/ComboLock/2017-4-11
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RCA Double Deuce[edit]
Experiment is going to test how well RCA works (again). For this experiment I will be using 2 primers, the LLRC***, which targets the + strand (that is the padlock/circular amplicon itself) and primer12RC, which targets the (-) strand. This should create extra amplification and
Protocol[edit]
- Rolling Circle Amplification
- Prepare master mix according to table below
- Add 15 uL master mix to each tube
- Samples "A-C" get + master mixes; samples "D-F" get - master mixes
- Add 1 uM RCA primer and to new reaction tubes according to following rules
- "A,D" samples get 2 uL each LLRC & primer12RC
- "B,E" samples get 2 uL each LLRC*** & primer12RC
- "C,F" samples get 4 uL LLRC***
- Heat reactions up to 95C for 5 min
- Cool to 55C and incubate for 15 minutes
- Cool to 30C and add enzyme
- Incubate for 3 hours at 30C
- Incubate for 65 minutes at 20C to denature
Reagent | Single Rxn (uL) | Master Mix X+ (3.2X) | Master Mix X- (3.2X) | Master Mix Y+ (3.2X) | Master Mix Y- (3.2X) | Joker |
Enzyme | NA | NEB | NEB | Thermo | Thermo | NEB |
Circular Product (1X-1nM) | 2 | 6.4 | 0 | 6.4 | 0 | 2 |
Linear Product (1X) | 2 | 0 | 6.4 | 0 | 6.4 | 0 |
RCA Primer (1 uM each) | 2 | 0 | 0 | 0 | 0 | 2*** |
dNTP (1 mM) | 5 | 16 | 16 | 16 | 16 | 5 |
10X Buffer | 2 | 6.4 | 6.4 | 6.4 | 6.4 | 2 |
Phi29 | 1 | 0 | 0 | 0 | 0 | 1 |
BSA (10 mg/mL) | 0.4 | 1.28 | 1.28 | 0 | 0 | 0.4 |
nfH2O | 5.6 | 17.92 | 17.92 | 19.2 | 19.2 | 7.6 |
Total | 20 | 48 | 48 | 48 | 48 | 20 |
Continued tomorrow