Cloning with LambdaGEM-12
lippay at vaxd.gat.com
Wed Nov 2 19:41:04 EST 1994
In article <devitta-0211941125380001 at bcs93.bham.ac.uk>,
devitta at uk.ac.birmingham (Chutney) wrote:
> LambdaGEM-12 Xho I half-site arms cloning system.
> In this system LambdaGEM-12 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
> been partially filled-in with dATP and dGTP.
> Using this cloning system (from Promega) I have been obtaining
> unusually high levels of background when vector DNA is ligated and
> packaged in the absence of insert genomic DNA. Typically 20,000
> pfu/microgram of vector DNA.
> Has anybody else had similar problems?? I would appreciate any advice
I have used the lambda Gem 11 vector from Promega which has the same
cloning strategy as Gem 12. When ligating the arms without insert the
background was zero. I did this twice to make sure I did not make a mistake
the first time. With insert the ligation was very effecient. I would think
maybe you have a bad batch of vector.
General Atomic Biosciences Div.
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