RACE

vilimf01 at mcrcr6.med.nyu.edu vilimf01 at mcrcr6.med.nyu.edu
Mon Apr 1 20:58:03 EST 1996


I have been in your situation, having been involved with the cloning
of several neuropeptide precursors.  You dont mention the source and
expected abundance of your message, so I can only provide some general
comments.  As far as the race goes, a southern on your pcr products
with some possible internal (even degenerate) oligo is a must to
identify the real from the artifact.  Same oligo can then be used to
screen t/a subclones of a gel purified positive band.  Offhand, if you
dont have more peptide sequence than AAPLW, you dont have much of a
chance with race (degeneracy=384, 15mer).  Asider  degeneracy and
length, you would tend to favor the amplification of the petide copy
closest to the 5' end making the identification by multiple copies
unlikely. 
     As far as alternative approaches go, if you have an antibody, you
could try expression cloning.  If you know the tissue distribution,
and can acess them, you could try differential display.  If the
precursor is known in another species, you could guess at conserved
regions and do PCR, or low stringency screens.  Probably, there are
many other approaches, the best one depends on your particular
situation.
			Usual Disclaimers		Sven



In article <31612B04.41C6 at zi.biologie.uni-muenchen.de>, Klaus Salger <salger at zi.biologie.uni-muenchen.de> writes:
> Dear netters,
> 
> 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  
>   Maria



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