background hybridization

Tom and Natarajan N490047 at UNIVSCVM.CSD.SCAROLINA.EDU
Wed Jul 7 10:52:49 EST 1993


We have been screening libraries constructed in lambda ZAP II at the XhoI
site using a two base fill-in.  Once we have identified initial clones, we
have been trying to use these inserts as probes to walk.  The problem is that
we cannot remove all of the vector sequences and have been getting a lot of
background hybridization.  We believe that this is due to the G/C rich element
proximal to the KpnI site of pBSSK-.  We have tried extremely stringent washing
 conditions and have still had limited success at eliminating background
hybridization.  Also our inserts are very G/C rich which could contribute.
We are currently having to resort to labelling aplified products made from
internal primers, but this is a pain in the ****!  Any suggestions?

Tom and Natarajan



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