Probing phage library

Steven Goldberg goldberg at bms.com
Fri Jun 16 15:54:39 EST 1995


I have been attempting to isolate a clone from Clontech's
pig liver cDNA library (in lambda gt11). This should be
relatively straightforward as the identical clone was
previously obtained from the same library by another
group.  In fact, the sequence was published so I was able
to make specific oligonucleotides to the desired gene.
I have screened ca. 1,000,000 independent clones without
a hint of a positive plaque, so I hope someone may be able
to give me some direction.
 I am following standard protocols for plaque lifts using 
nylon filters.  I am hybridizing according to Clontech's
manual (6x SSPe/1X Denhardt's/0.25% SDS/100 ug/ml hetero-
logous DNA at Tm-20).  Washes are at Tm-30 in 2X SSC/0.05%
SDS for 1 hr followed by 15 min in the same buffer.
 What concerns me is that I don't even see a background of
plaques at this relatively low stringency.  I have been
end labeling 25- and 30-mer oligos using T4 kinase and 
purifying using Clontech's spin columns, which usually 
gives me good results.  In fact, I have tried to probe 
a lift of their chicken ovalbumin cDNA clone control under
similar conditions and again, not a hint of background,
much less a positive plaque. My guess is that for some
reason my system is not sensitive enough.  Any ideas/
comments/suggestions???   





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