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

5' RACE PCR


5' RACE PCR

5' RACE PCR Protocol

1. First strand cDNA synthesis:
- Reaction set up:

cDNA synthesis buffer 4 ul
dNTP mixture 2 ul
gene specific primer 1 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

- Mix well and spin down briefly.
- Incubate at 55 C for 1 hour.
- Heat termination of reaction at 65 C for 10 min.
- Spin down briefly.

2. Purification of cDNA using PCR product purification column of your choice.

3. Tailing reaction of cDNA
- Reaction set up:

Purified cDNA products 19 ul
10x reaction buffer 2.5 ul
2 mM dATP 2.5 ul
--------------------------------------------------------------
Total 24 ul

- Incubate at 94 C for 3 min.
- Stand on ice for 1 hour.
- Spin down briefly and add 1 ul of terminal transferase (10 unit/ul).
- Mix and incubate at 37 C for 20 min.
- Incubate at 70 C for 10 min to inactivate the terminal transferase enzyme.
- Spin down and place reaction tube on ice.

4. PCR amplification of dA-tailed cDNA
- Reaction set up:

dA-tailed cDNA 5 ul
oligo-dT anchor primer 1 ul
gene specific primer 2 1 ul
dNTP mixture 1 ul
Taq polymerase 0.5 ul
10x PCR reaction buffer 5 ul
ddH2O 36.5 ul
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Total 50 ul

Negative control: add water 41.5ul without adding dA-tailed cDNA

- Mix well and spin down briefly.
- Start PCR

5. 5' nested PCR using gene specific primer 3

- Dilute 1000 times of the first PCR products for nested PCR.
- PCR reaction set up:

Diluted PCR products 1 ul
PCR anchor primer 1 ul
gene specific primer 3 1 ul
dNTP mixture 1 ul
Taq polymerase 0.5 ul
10x PCR reaction buffer 5 ul
ddH2O 40.5 ul
--------------------------------------------------------------
Total 50 ul

Negative control: no diluted PCR products.

Note:

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

Last update 15-Nov-2007, Rating Fair of 2 votes.

Write your comment

By Deepankar Gahloth on 25-Jun-2008
No concentartion and protocol for removing excess dNTPs and primers is not given, which is most important aspect for the successful completion of 5'RACE.
Rating: Fair

Reply
By S. L on 15-Nov-2007
We do not have concentrations here, just volumes! So it cannot help much. Also, number of cycles?
Rating: Fair

Reply


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