Daniel:Protocols/RCA
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Circularization and Rolling Circle Amplification
Protocol page for basic circularization of padlock probes and rolling circle amplification of the finished product. Includes exonuclease digestion to get rid of
Protocol
- Circularization
- Prepare (N+1)X uL Phusion mix (N samples)
- Add 20 uL Phusion mix to 20 uL of reaction suspended in 1X buffer
- Incubate at 60C for 2 hours
- Heat kill the reaction by incubating for 2 minutes at 94C
- Exonuclease Digestion
- Combine equal molar amounts of Exonuclease I (20U/uL) and Exonuclease III (100U/uL)
- Add 2 uL of mix to each reaction
- Incubate for 30 minutes to 2 hours at 37C
- Heat kill by incubating for 5 minutes at 94C
- Rolling Circle Amplification
- Prepare (N+1)X master mix according to table below
- Add 15 uL master mix to each tube
- Add 5 uL sample to correct reaction tubes
- Incubate at 37C for 3 hours
- Heat kill by incubating at 65C for 10 minutes
Reagent | Stock Conc | Final Amount | 1x Vol (uL) |
NAD+ | 5 mM | 40 nmol | 8 |
dNTP | 1 mM | 600 pmol | 0.6 |
Betaine | 5 M | 15 umol | 3 |
10X AmpLigase Buffer | 10X | 1X | 2 |
Amp Ligase | 5 U/uL | 10 U | 2 |
Phusion HF DNA Polymerase | 2000 U/mL | 6.4U | 3.2 |
nf H2O | 1.2 | ||
Total | 20 |
Reagent | Stock Concentration | Final Conc/Amt | uL Added in 20 uL Rxn |
Template | NA | NA | 5 |
RCA Primer | 10 uM | 2 uM | 4 |
dNTP | 1 mM | 50 uM | 1 |
10X Buffer | 10X | 1X | 2 |
Phi29 (NEB) | 10 U/uL | 10 U | 1 |
nfH2O | NA | NA | 7 |
Total | 20 |