Daniel:Notebook/ComboLock/2017-3-26
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RCA Test (Started March 24)[edit]
Linear Product Production-Bglii (Started Yesterday)[edit]
- 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
File:2017-03-26-LinearProductProduction-Bglii.png
That's the right one. However, there is a ton of primer (bottom of the gel), so I'll run a ssDNA column to fix.
- ssDNA column
- Add 50 uL Binding Buffer to the sample; mix well
- Transfer to IIC Column and centrifuge at 14000 rpm for 1 minute; SAVE THE FLOW THROUGH
- Add 70 uL 100% EtOH to flow through; mix well
- Transfer to IC Column and centrifuge at 14000 rpm for 1 minute; discard flow through
- Add 400 uL Prep Buffer and centrifuge at 14000 rpm for 1 minute; discard flow through
- Add 700 uL Wash Buffer and centrifuge at 14000 rpm for 1 minute; discard flow through
- Add 400 uL Wash Buffer and centrifuge at 14000 rpm for 1 minute; discard flow through
- Centrifuge empty column at 14000 rpm for 2 minutes
- Transfer to empty 1.5mL centrifuge tube (low bind)
- Add 20 uL nfH2O and centrifuge at 14000 rpm for 1 minute
- Rolling Circle Amplification
- Prepare master mix according to table below
- Add 16.5 uL master mix to each tube
- Samples "A-C" get + master mixes; samples "D-F" get - master mixes
- Add 2.5 uL 10 uM RCA primer and to new reaction tubes according to following rules
- "A,D" samples get LLRC
- "B,E" samples get LLRC-v03 (Mono-phosphorothiorate)
- "C,F" 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
RCA Protocol[edit]
Reagent | Single Rxn (uL) | Master Mix X+ (9.2X) | Master Mix X- (9.2X) | Master Mix Y+ (3.2X) | Master Mix Y- (3.2X) | Master Mix Z+ (3.2X) | Master Mix Z- (3.2X) |
Enzyme | NA | NEB | NEB | Thermo | Thermo | Epi (1:10) | Epi (1:10) |
Circular Product (1X) | 2 | 18.4 | 0 | 6.4 | 0 | 6.4 | 0 |
Linear Product (1X) | 2 | 0 | 18.4 | 0 | 6.4 | 0 | 6.4 |
RCA Primer (100 uM) | 2.5 | 23 | 23 | 8 | 8 | 8 | 8 |
dNTP (1 mM) | 5 | 46 | 46 | 16 | 16 | 16 | 16 |
10X Buffer | 2 | 18.4 | 18.4 | 6.4 | 6.4 | 6.4 | 6.4 |
Phi29 | 1 | 0 | 0 | 0 | 0 | 0 | 0 |
BSA (10 mg/mL) | 0.4 | 3.68 | 3.68 | 0 | 0 | 0 | 0 |
nfH2O | 8.1 | 65.32 | 65.32 | 24 | 24 | 24 | 24 |
Total | 20 | 117.8 | 117.8 | 59.52 | 59.52 | 59.52 | 59.52 |
Sample | Master Mix | Enzyme | Incubation Time (hrs) |
X (A-F) | X | NEB | 3 |
Y (A-F) | Y | Thermo | 3 |
Z (A-F) | Z | Epi (1:10) | 3 |
P (A-F) | X | NEB | 6 |
Q (A-F) | X | NEB | 12 |
Continued tomorrow