Cloning with LambdaGEM-12

[Eric Lippay]

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
> available.

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.

Eric Lippay
General Atomic Biosciences Div.