Technique / Molecular Biology / PCR / 5' and 3' RACE

3' RACE PCR


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
-------------------------------------------------------------
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.

Write your comment

By T to the EO on 12-Feb-2008
Nature methods vol.2 no.8 august 2005 (629-630)
Complete en detailed protocol
Rating: Fair

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By Susan on 03-Nov-2007
I really needed this illustration. It really helps a lot understand the principle.
But How do you know where the gene specific 5' end is separated from the polyA tail? I work in a high AAA and TTT organism so it might be tricky...
Rating: Excellent!

Reply
By Memo Jenn on 09-May-2007
more explanation should be given regarding how the protocols work. A complete protocols for the complete experiment from mRNA extraction to gel electrophoresis process can be given especially about plant genome because I am interested with that experiment.
Rating: Fair

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