3' RACE PCR

3' RACE PCR

Protocol:

1. First strand cDNA synthesis:
- Reaction set up (total volume 20 ul)

cDNA synthesis buffer 4 ul
dNTP mixture 2 ul
oligo-dT anchor primer 1 ul
total RNA 1 ul
AMV reverse transcriptase 1 ul
RNasin (RNase inhibitor) 0.5 ul
0.1M DTT 1 ul
ddH2O 9.5 ul
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Total 20 ul


- Spin down briefly and incubate at 55 C for 1 hour.
- Stop reaction: 65 C for 10 min.
- Briefly spin down reaction mixture.

2. RACE PCR amplification of cDNA:
- Reaction set up:

cDNA 1 ul
PCR anchor primer 1 ul
gene specific primer 1 ul
dNTP mixture 1 ul
Taq polymerase 0.5 ul
10x PCR reaction buffer 5 ul
ddH2O 40.5 ul
-----------------------------------------------------------
Total 50 ul

For negative control: no cDNA template, add 41.5 ul ddH2O.

- Mix well and spin down briefly.
- PCR amplification.
- Perform PCR products analysis on 1.5% agarose gel.

Note:

see RACE PCR illustration for details of PCR anchor primer, gene specific primer design and general overview.

Last update 09-May-2007, Rating Good of 3 votes.