salger at zi.biologie.uni-muenchen.de
Tue Apr 2 08:26:28 EST 1996
I want to clone a cDNA coding for a peptidic family called AALPW.I think
these family of peptides are codified in the same cDNA having a kind of
spacer amino acid sequence among them. I used a set of degenerate oligos
15mer and a T18-Not primer. I did 3'RACE and the results are not very
succesfully. First, I obtained a lot of smear, where one can see some
more defined bands. But I can not find a good conditions on PCR and get
a sharp band for cloning.A second problem, In any case, because the
pattern was always quite constant on different PCR conditions, I cloned
the bands that I obtained, but on no one cloned I could find the
expected cluster of peptides.
My PCR conditions were from 60 C to 43 C. The theorical annealing
temterature of the specific oligo is 46 C. I did touch down with
different ranges of tempetature. For example, from 60 to 50 in 4 steps;
from 55 to 48, from 53 to 43. In general, I do 3 cycles on the highest
temperatures and 30 cycles on the last annealing temperature.
How I can make a better 3' RACE?
How I can clone these family of peptides?
Thanks in advance
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