Problems in a fish genomic library construction

ROBERT SAUNDERS rdcsaunders at its.dundee.ac.uk
Fri Jun 21 02:33:43 EST 1996


In article <Pine.OSF.3.91.960620163259.8499B-100000 at Galeon.uca.es> LUIS BEJARANO ARDURA <lba at Galeon.uca.es> writes:
>From: LUIS BEJARANO ARDURA <lba at Galeon.uca.es>
>Subject: Problems in a fish genomic library construction
>Date: Thu, 20 Jun 1996 16:44:43 +0200

>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.

> 

I would advise using sucrose gradient centrifugation to prepare the vector 
arms, and for size selecting the partially digested genomic DNA.  There is a 
good description in Sambrook et al.

Robert




More information about the Methods mailing list