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Star Republic: Guide for Biologists

3'-RACE

Edited by Chang Zhu
3'-RACE (Rapid Amplification of mRNA ends by PCR) clones 3'-ends of mRNA. Database search can sometimes be used to find mRNA ends. See EST assembler for more information.

Synthesis of the cDNA
1. Place 1-10 mg of total RNA (or 0.1-1 mg polyA RNA) in a RNAse-free eppendorf. Adjust the volume to 10 ml with DEPC-treated RNase-free water.

2. Heat the RNA sample to 70 C for 5 minutes to denature secondary structures, immediately cool the sample on ice.

3. Add the following reaction mix:

10X AMV Reverse transcriptase buffer 2 ml
10 mM dNTP 2 ml
RNasin Inhibitor 1 ml
T17 Adapter primer (200 ng/ml ) 2 ml
AMV Reverse transcriptase 1 ml

T17 adapter primer: 5'-GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT-3'

4. Mix, and incubate at 42 C for 1-2 hours.

PCR amplification of the 3'-ends
5. Take 1 ml of the 1:10, 1:30, and 1:100 dilution of the reverse transcription mix, set up standard 50 ml PCR reactions. Use the gene specific primer as the forward primer and the T17 adapter primer as the reverse primer.

6. Run PCR reactions for 30-40 cycles.

7. Checking PCR products by running an aliquot (5-10 ml) on the argarose gel.

Cloning the RACE products
8. RACE products can be cloned directly. Or isolate the most prominent bands, digest with restriction enzymes recognizing sites in the T17 adapter and the gene specific primer, and clone into a vector.