3' R apid A mplification of c DNA E nds (RACE ) PCR

This technique is used to obtain the 3'end of a cDNA, it requires some sequence information internal to the mRNA under study. The sequence information obtained from this technique can be utilised to obtain full length cDNA clones using the 5'RACE technique.

The method I've used is loosely based on that given in 'PCR protocols: A guide to methods and applications', Academic Press Inc.

The protocol given uses SuperScript II reverse transcriptase (Life Technologies) and Taq polymerase from Boehringer Mannheim. You will need to adjust the reagents according to the enzymes you use.

Reagents:

Sterile water (high purity, RNase-free)

5 x SuperScript RTase buffer (Life Technologies)

100mM DTT

2.5mM dNTPs

RNase inhibitor (Pharmacia)

SuperScript RTase (Life Technologies)

10 x PCR buffer (Boehringer Mannheim, contains 15mM MgCl2 )

500mM dNTPs

Mineral oil

RNA : Both total and polyA RNA are suitable for this technique. I recently compared the two and found the polyA RNA reaction to have the edge, but only just. I do recommend using polyA RNA for 5'RACE though.

Primers:-

T17 Adapter primer (T17AP): GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT

Adapter primer (AP): GACTCGAGTCGACATCG

Gene specific primer: Designed from known sequence, ideally it should an 18-22-mer with an annealing temperature of around 56℃, where a G/C = 4℃ and an A/T = 2℃.

Protocol

1. Place approx. 1ug of total RNA (~10ng polyA RNA) in an RNase-free eppendorf, make up to 10μl with pure water. Add 1μl of T17AP (0.5ug/ul).

2. Heat this to 70℃ for 10 minutes, then place on ice for 5 minutes.

3. Spin pulse the tube and then add the following reagents:

4μl 5X SuperScript RTase buffer

2μl 100mM DTT

1μl 2.5mM dNTPs

1μl RNase inhibitor

1μl SuperScript RTase.