Cloning problems with LambdaGEM-12

Fiona Cassidy f.a.cassidy at uk.ac.birmingham
Wed Nov 9 07:16:03 EST 1994


LambdaGEM-12 Xho I half-site arms cloning system.

In this system lambdaGEM-12 DNA has been digested with Xho I, partially
filled in with dTTP and dCTP, and dephosphorylated.  This allows it to
ligate specifically with Mbo I- or Sau 3A I-digested genomic DNA which has
in turn been partially filled in with dATP and dGTP.

Using this lambdaGEM-12 cloning system (from Promega) I have been
obtaining unusually high levels of background, where vector DNA has been
ligated and packaged in the absence of insert genomic DNA.  These were
typically
20,000 pfu/microgram of vector DNA.  The lot numbers of the kits used were
188802 and 3242023. I was also using Promega's packagene extracts.
   Has anybody else had similar problems?  Any advice would be greatly
appreciated.



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