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- 10U RNA guard (Pharmacia)
- 10U DNase I
- 10mM Tris-HCl buffer, pH 8.3
- 50mM KCl
- 1.5mM MgCl2
- 20ug Total RNA
- Add RNA water to 50ul
- 30oC, 30Min.
- 10ul RNA Water
- 1ul DNA-free total RNA
- 4ul 5Xreverse transcription buffer
- 2ul 0.1M DTT
- 1ul 10mM dNTP stock
- 1ul Reverse transcriptase
- 1ul T12NA, T12NT, T12NG or T12NC (for separate reactions)
- 37oC, 60min.
- 15ul 33P-dATP
- 50ul 10XPCR buffer
- 5ul 0.2mM dNTP
- 10ul Taq polymerase (5u/ul)
- 20ul H2O
- 2ul RT PCR product
- 12ul PCR reaction stock&
- 45ul H2O
- 3ul oligo-dTplus primers(T12NA, T12NT, T12NG or T12NC) in the repective tubes
- 94oC, 5min., link to 40 cycles of:
- 94oC, 30sec.
- 40oC, 2min.
- 72oC, 30sec.
- Link to 72oC, 5min., hold at 4oC
- 30g urea
- 6ml 5XTBE buffer
- 9ml 38:2 acrylamide
- 23ml water, mix gently
- Add 120 ul 20% APS and 60ul TEMED
- 30ul Water
- 1ul DNA fragment purified from gel
- 2ul 2mM dNTP
- 1ul oligo-T12NX primer (corresponding the primary reaction)
- 1ul andom primer(corresponding the primary reaction
- 5ul 5XPCR buffer
- 10ul 50% glycerol
- Add 1 drop of oil.
This step can also be carried out by using a PCR stock solution to reduce the pepetting. To make the PCR stock, mix:
- 840ul pure water
- 54ul 2mM dNTP
- 140ul 10XPCR buffer
- 280ul 50% glycerol
- For a 50ul reaction, use:
- 46.5ul the stock
- 1ul DNA fragment
- 1ul 3'primer
- 1ul 5' primer
Note: To have a pure PCR product, it will help to run the secondary PCR product together with the original in a sequencing gel and cut out the band again.