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
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:
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.