Problems in a fish genomic library construction
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
>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.
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