In article <54j7mi$8nc at pulp.ucs.ualberta.ca>, plagali at gpu2.srv.ualberta.ca
(Pamela Lagali) wrote:
> Hello,
>> 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
> arms. In one of my primary screens, a very faint signal was detected for
> one plate, which may indicate a positive phage plaque. I performed a
> secondary screen and see signals of the same low intensity for a few
> plaques, though much fewer "positive" signals than I would expect for a
> secondary screen. I would like to know if these are in fact "positives",
> or if the low signal intensity is indicative of non-specific binding of my
> probe (i.e. merely background). Does anyone have a tested protocol for
> screening libraries using oligonucleotides as probes which gives low
> background and strong signals for positive phage plaques? Are false
> positives common? What protocol do you use to label the oligo?
>> Any assistance would be greatly appreciated.
>> Thanks,
>> Pam
>>> Pamela Lagali
> Department of Cell Biology
> University of Alberta
> Edmonton, Alberta, Canada
> T6G 0X6
> ph: (403) 492-7407
Hi Pamela
I had the same problem when I was trying to screen my library which was
made of cloned PCR products.
Generally, the signal of oligonucleotide probe is not very strong enough
to cause background problems. I don't know what protocol (esp. hybing mix
and washing condition) you are using, but in my case I could get very
strong signal without any background by minimizing SDS content down to 0.1
%.
Good luck.
