In article <54j7mi$8nc at pulp.ucs.ualberta.ca>, plagali at gpu2.srv.ualberta.ca
(Pamela Lagali) wrote:
>> I am trying to use a gamma 32-P end-labelled synthetic oligonucletide
> (30-mer) as a probe for screening a cDNA library cloned into lambda phage
Pam: If you only have a faint signal I would guess its just a false
positive. It is important to screen duplicate filters, especially with
oligonucleotide probes. I have found that true positives will be very
dark compared to background (unless you suspect you don't have complete
homology between your probe and target).
I like to use end labeling with terminal transferase plus alpha-dATP or
-dCTP. It seems to be much less sensitive to contaminants than kinase +
gamma-dNTP, but this is a personal preference. If there is any way you
can get a PCR product of the gene you want to isolate (or something
related), this is the best probe to use.