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oligonucleotides as probes

Pamela Lagali plagali at gpu2.srv.ualberta.ca
Tue Oct 22 14:35:46 EST 1996


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.



Pamela Lagali
Department of Cell Biology
University of Alberta
Edmonton, Alberta, Canada
T6G 0X6
ph: (403) 492-7407

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