cDNA cloning problem - No product in PCR (Sep/04/2005 )

i would like to clone an unknown hormone gene or cDNA.
i have tried to do it using 3'/5' race using primers that i have designed acording to an sequence alignment. however i failed - haven't seen any product even if i do 50 cycles. i think that it is because the gene expression is too low.
i checked my primers and they are fine.
before i try to do a southern blot and mini genomic libarary (using a radioactive probe), can i clone this gene (or part of it) using a genomic DNA as a template by regular pcr method?.
thanx for any other idea

Hi gall,

If you want to clone the gene (cDNA) for constructing an expression vector you cannot use genomic DNA as template.

How big is the expected size of your gene (cDNA sequence)? How did you do the first strand cDNA synthesis? How did you decide the primer sequences? Those are questions we need to know before we can troubleshoot your problem.

my sequence is not so big the gene - 2000bp and the cDNA 500-600bp. according to the sequence alighnment from other relative organisms my pcr product should be 200-400 bp using the primers i desighned. the gene is very conservative. 80-90% identity between its orgenisms from the same order (due to the genebank).
i synthesized the cDNA using a amv enzyme and also with abgene kit.
for the pcr i used diluted and undiluted cDNA and also poly a tailed cDNA for 5' race as template. any other ideas???

Do I understand it correctly that your template for the PCR is genomic DNA?

If so, I generally find that PCRs with 5-10% DMSO work best, but you have to check if your Taq pol works with DMSO (Promega's doesn't).

i probably didn't explain myself correctly. what i'm tryning to do is to clone part of the hormone from genomic DNA and also from cDNA pool as a template. just to see any fragment for further cloning and sequencing not for expression. i use specific primers. the question actualy is what is the ptobability to have a specific fragment with genomic DNA as a template.
about my pcr - the positive control work fine ... why to use DMSO??

was your positive control genomic DNA as well?

DMSO relaxes socondary structure in genomic DNA and therefore makes the denaturing easier.

You could also try and do a Temperature gradient PCR, if you can get your hands on a PCR machine that has the ability to do that. (This is a machine which gives a different annealing temperature in each column of wells).