help concerning lambda library screening with DIG system

Nobody nobody at hgmp.mrc.ac.uk
Wed Jan 31 12:44:20 EST 2001


Hi,

I have a question to the community concerning the reuseability of plaque
lifts from a lambda genomic library (A. t.) for hybridisation with the
DIG system. When we screened it first, it worked perfectly, giving about
20 positives per plate (50000 cfu). However, when we stripped the
library (10 min boiling 0.1% SDS in dH20) and rehybridised again with a
different probe (which is working well, we checked this), no signal at
all was obtained. Is it possible that we lost practically the whole
plaque DNA during stripping (this is what one of us believes), or is it
rather due to the fact that plaque lifts should be stored different than
we did (4=B0 several months), should be stripped from the antibody-AP
conjugate (it is an phosphatase, after all...) or even due to the fact
that reprobing is not good with this system? Concerning Southern blots
and some Northern blots, we are highly satisfied with the DIG system,
but library screening tends to be a pain sometimes.

thanks,

Harald

(if you answer directly to me, please use hsigmund at edv2.boku.ac.at)


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