Daniel:Notebook/ComboLock/2017-3-25
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RCA Test
This experiment is to test the effectiveness of RCA so that I can use the most efficient method of amplification for the cell-based experiments. This builds on the T4 experiments and the circular product production. I will be using that circular product to analyze the effectiveness of RCA.
Linear Product Production (Continued from yesterday)
- TBE gel
- Mix 64 uL TBE and 16 uL 6X dye
- Aliquot 10 uL on to parafilm per sample/ladder
- Add 2 uL reaction, 1 uL (10 uM) controls, and 1.5 uL ladder to appropriate aliquot File:GelLanes-20170325-LinearProduct.png
- Add 10 uL mix to gel lanes
- Run gel for 24 minutes at 230V
- Stain with 2 uL SYBR gold for 3 minutes
- Rinse and image in gel doc
RCA Protocol
- Rolling Circle Amplification
- Prepare master mix according to table below
- Add 16.5 uL master mix to each tube
- Add 2.5 uL 10 uM RCA primer and to new reaction tubes according to following rules
- "A" samples get LLRC
- "B" samples get LLRC-v03 (Mono-phosphorothiorate)
- "B" samples get LLRC-v04 (Tri-phosphorothiorate)
- Heat reactions up to 95C for 5 min
- Cool to 55C and incubate for 15 minutes
- Cool to 30C and add enzyme
- Incubate at 30C for length of time according to table below
- Incubate at 65C for 10 minutes;Hold at 10C until next step
Reagent | Single Rxn (uL) | Master Mix X+ (6.2X) | Master Mix X- (6.2X) | Master Mix Y+ (3.2X) | Master Mix Y- (3.2X) | Master Mix Z+ (3.2X) | Master Mix Z- (3.2X) | Master Mix P+ (3.2X) | Master Mix P- (3.2X) | Master Mix Q+ (3.2X) | Master Mix Q- (3.2X) |
Enzyme | NA | NEB | NEB | Thermo | Thermo | Epi (1:10) | Epi (1:10) | NEB | NEB | NEB | NEB |
Circular Product (1X) | 4 | 24.8 | 0 | 12.8 | 0 | 12.8 | 0 | 12.8 | 0 | 12.8 | 0 |
Linear Product (1X) | 4 | 0 | 24.8 | 0 | 12.8 | 0 | 12.8 | 0 | 12.8 | 0 | 12.8 |
RCA Primer (10 uM) | 2.5 | 15.5 | 15.5 | 8 | 8 | 8 | 8 | 8 | 8 | 8 | 8 |
dNTP (1 mM) | 2 | 12.4 | 12.4 | 6.4 | 6.4 | 6.4 | 6.4 | 6.4 | 6.4 | 6.4 | 6.4 |
10X Buffer | 2 | 12.4 | 12.4 | 6.4 | 6.4 | 6.4 | 6.4 | 6.4 | 6.4 | 6.4 | 6.4 |
Phi29 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
BSA (10 mg/mL) | 0.4 | 2.48 | 2.48 | 0 | 0 | 0 | 0 | 1.28 | 1.28 | 1.28 | 1.28 |
nfH2O | 8.1 | 50.22 | 50.22 | 25.92 | 25.92 | 25.92 | 25.92 | 25.92 | 25.92 | 25.92 | 25.92 |
Total | 20 | 117.8 | 117.8 | 59.52 | 59.52 | 59.52 | 59.52 | 60.8 | 60.8 | 60.8 | 60.8 |
Sample | Enzyme | Incubation Time (hrs) |
X (ABC) | NEB | 3 |
Y (ABC) | Thermo | 3 |
Z (ABC) | Epi (1:10) | 3 |
P (ABC) | NEB | 6 |
Q (ABC) | NEB | 12 |