EricChu:RepliPHI MDA protocol (20ul reaction)
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RephiPHI MDA Protocol (20ul reaction)[edit]
- Start with 1.0ul λ DNA (2.5ng/ul) in PCR tube
- Add 1.5ul ALS, incubate at room temp 3mins
- Add 1.5ul NS, put on ice immediately
- Create master mix (w/ 1% Tween20)
- 2ul 10x buffer
- 5ul 200uM N6 primers
- 0.8ul 25mM dNTPs
- 2ul Phi29 polymerase
- 2ul 8% Tween20
- 4.2ul H20
- Add master mix to PCR tube
- Add to thermocycler for 10 hours at 30⁰C and keep at 4 ⁰C
- Use PCR purification kit to collect DNA
- Measure DNA concentration with NanoDrop
ALS: 69.5ul H20 + 10ul DTT(1M) + 8ul KOH (5M) + 12.5ul 8% Tween20
NS: 87.5ul NS stock + 12.5ul 8% Tween20
where NS stock: 4ml 1MHCl and 6ml 1MTrisHCl