5' RACE-Tail cDNA with dC, or dT?
anmab2 at orion.alaska.edu
Thu Oct 5 00:50:28 EST 1995
I have some limited experience with 5' RACE, and when I performed it, I
tailed my first strand cDNA with dATP. Perhaps the concern here is two
fold...1). the higher Tm of a poly dC tail relative to a poly dA tail.
2). the poly dA primer/adaptor can be utilized for both 5' and 3' RACE
Regarding the use of your degenerate primer for the initial PCR
reaction...when I've performed this, I've always primed with one
gene-specific primer during first-strand synthesis and carried out the
PCR with a second gene-specific oligo. In theory, you should be able to
carry out your PCR with the same oligo(s) you primed your mRNA with. In
fact, in CPMB's protocol for 5'RACE, they have you do two seperate PCR
amplifications: first with the oligo used to prime the mRNA and then re
amplify with a second gene specific oligo.
The only problem I can forsee with doing the PCR with the same oligo
used to prime the mRNA is one of specificity. If your primer is
degenerate, you will get more "false" priming to begin with, which the
use of a second gene-specific oligo would help to minimize. So if you
only use the single set of degenerate primers, you will probably want to
subclone and do colony screening on your transformants prior to
sequencing to take care of that problem.
Hope this helps.....
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