Daniel:Notebook/ComboLock/2017-2-28
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RCA + Circularization Test (Started Yesterday)
Protocol
- ssQubit
- Mix together 796 uL nfH2O and 4 uL dye (200:1 ratio)
- Add 190 uL mix and 10 uL S1 or S2 to standard tubes
- Add 199 uL mix and 1 uL sample to sample tubes
- Measure in Qubit; use new run and calibrate with standards
- Column Purification-Qiaquick
- Add 5X PB (binding buffer) to sample
- Load sample onto column and spin for 1 minute at 14000 rpm; discard flow through
- Add 750 uL PE (wash buffer) to column and spin for 1 minute at 14000 rpm; discard flow through
- Dry spin column for 1 minute at 14000 rpm; discard flow through
- Let stand with cover open in fume hood for ~5 minutes
- Transfer column to a new 1.5 mL eppendorf tube
- Add ≥30 uL EB to column
- Let stand 1 minute
- Spin for 1 minute at 14000 rpm
- Rolling Circle Amplification
- Prepare master mixes with following enzyme layout
- Add 14 uL master mix to each tube
- Add 5 uL sample to new reaction tubes according to following table
- Incubate for 30 minutes at 75C
- Lower temperature to 30C
- Add 1 uL Phi29 polymerase and incubate 3 hours at 30C
- Heat kill enzyme with 10 minutes at 65C
- Hold at 4C overnight
Samples | Condition |
Sample X (1-2,ABC) | NEB |
Sample Y (1-2,ABC) | Thermo Fisher |
Sample Z (1-2,ABC) | Epicenter |
A Samples (1-2, XYZ) | Normal Primer |
B Samples (1-2, XYZ) | Phosphorothiorate Primer |
C Samples (1-2, XYZ) | Column Purified Product |
Reagent | Single Rxn Vol | MM-X (NEB) uL | MM-Y (TF) uL | MM-Z (Epi) uL |
Template | 5 | 0 | 0 | 0 |
RCA Primer (10 uM) | 2.5 | 15.25 | 15.25 | 15.25 |
dNTP (1 mM) | 5 | 30.5 | 30.5 | 30.5 |
10X Buffer | 2 | 12.2 | 12.2 | 12.2 |
Phi29 | 1 | 0 | 0 | 0 |
BSA (10 mg/mL) | 0.4 | 2.44 | 0 | 0 |
nfH2O | 4.1 | 25.01 | 27.45 | 27.45 |
Total | 20 | 85.4 | 85.4 | 85.4 |