Primer for 5' RACE - (Jun/07/2006 )
i've some questions concerning 5' RACE PCR, especially creating the primer using for the initial RT-reaction.
in a normal RT-reaction i use oligodT primer for amplifying cDNA from mRNA, cause the oligodT primes the polyA-tail.
for the 5' RACE it is recommended to do the RT-reaction with a gene specific primer. so i have to create a primer against my mRNA of interest, so that only cDNA of my gene is amplified?
right so far?
and how to do that? someone can give me an example?
no one any idea?
we use the SMART™ RACE cDNA Amplification Kit from Clontech to do the RACE PCRs (see attachment).
There, you use a 5`-CDS primer for the cDNA synthesis which is provided with the kit.
In the following RACE PCR (amplification of your desired product) you use a gene-specific primer. The specifications are: 23-28 nt, GC content = 50 - 70 %, high Tm. It has to be an antisense primer, so if your DNA sequence is:
gDNA: 5` aggtagtcgatcgactgctatggatctcgat 3`
you could chose this primer f.ex.: 5` atcgagatccatagcagtcgatcgac 3`.
It binds to the complementary strand of you DNA.