Problems in a fish genomic library construction

Carsten Hoege c.hoege at bham.ac.uk
Thu Jun 27 11:14:31 EST 1996


In article <Pine.OSF.3.91.960620163259.8499B-100000 at Galeon.uca.es>, LUIS BEJARANO ARDURA <lba at Galeon.uca.es> says:
>
>Hi netters,
>
>We want to make a fish genomic library in Lambda GEM 12 vector (Promega). 
>The Lambda GEM 12 arms have been cut with BamHI restriction enzyme and we 
>have eliminated the central stuffer (14 Kb) by electrophoresis in low 
>melting agarose gel. The genomic DNA has been cut with Sau3aI restriction 
>enzyme and dephosphorilated. Part of this genomic DNA has been 
>selectioned by electrophoresis low melting agarose gel about 10 - 20 Kb 
>size. We have tried all kind of combinations between Lambda GEM 12 BamHI 
>arms and our genomic Sau3aI DNA in the ligation reaction. We have also 
>tried several host strains like LE392, NM538 and KW251. What could we 
>check to increased our very low number of recombinants?. Many Thanks.
>
> 
>
Hi there!

I tried the GEM 12 kit as well. It was also hampered by low cloning efficiencies. 
The efficiencies of the packaging extracts vary enourmously. So it is very useful to
 carry out the positive control ligation and package the product. If the efficiency is too
 low, contact  Promega and complain.
The other problem might be the low purity of the partially digested DNA. Has the 
DNA been CsCl purified? Ligations with purified DNA will avour concatenated 
Lambda DNA that is packaged with much higher efficiency!
I used KW251, that was fine.
Hope that helps

Carsten



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