Daniel:Notebook/ComboLock/2017-1-30
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RCA Test (Started Monday)[edit]
Library Prep (Started Jan 26)[edit]
- Ethanol Precipitation
- Centrifuge for 30 minutes at 12500 rpm at 4C
- Remove supernatant and add 750 uL chilled 70% EtOH
- Centrifuge for 15 minutes at 12500 rpm at 4C
- Remove supernatant; dry in vacuum centrifuge for 15 minutes
- Resuspend in 20 uL H2O and consolidate samples
- Measure with Qbit
- Consolidate with samples from Thursday 1-26
DEJ20160126-CLv3-RCA02
Final Sample Matrix
Sample | Sample Amt | Pre/Post RCA | AmpR Index |
Sample 0 (AB) | 1 pmol | Pre | 21 |
Sample 0X (AB) | 1 pmol | Post | 25 |
Sample 2X (AB) | 10 fmol | Post | 26 |
Sample 4X (AB) | 100 amol | Post | 27 |
Sample 6C (AB) | 1 amol | Epi | 22 |
Sample 6D (AB) | 1 amol | Epi 1:10 | 23 |
RCA Timing Test (Started Friday)[edit]
Because of the results from Saturday, I think I want to try Epicentre again for as long as possible. I'll do 12 hours with (as usual) one negative control to save up on epicentre enzyme.
- Rolling Circle Amplification
- Prepare master mix according to table below
- Add 14 uL master mix to each tube
- Add sample and to new reaction tubes according to following table
- Incubate at 30C for 12 hours
- Incubate at 65C for 10 minutes;Hold at 10C until next step
Reagent | uL Added | Master Mix (4.2X) |
Template | 4 | NA |
RCA Primer (10 uM) | 2.5 | 10.5 |
dNTP (1 mM) | 2 | 8.4 |
10X Buffer | 2 | 8.4 |
Phi29 | 1 | NA |
nfH2O | 8.5 | 35.7 |
Total | 20 | 63 |
Continued tomorrow