How to separate many cDNA fragments in a plasmid?

Mikko Arvas Mikko.Arvas at vtt.fi
Mon Jan 20 04:10:26 EST 2003


Hi,

as a simple biologist I find the trivial problem below overwhelming. I 
would be extreamly grateful for any help.

I am sequencing a few thousand plasmids from a substraction cDNA library to 
find a few hundred novel unique genes of interest. The substractions and 
cloning have included 3 different restriction enzymes. If the ligations 
would have worked optimally every plasmid would contain only one cDNA 
fragment. As they have not about one third of the plasmids contain several 
different cDNA fragments separated by one of the 3 restriction sites. How 
can I separate these fragments from each other before the 
assembly/consensus sequence forming stage? Or is there a way to separate 
them some how automatically in GAP or a like program based on the presence 
of the restriction site? So far I have been using pherd for base calling 
and Staden after that.

Best regards,

Mikko Arvas
VTT Biotechnology - Research Scientist

e-mail:            mikko.arvas at vtt.fi
tel:                 +358-(0)9-456 5827
mobile:           +358-(0)50-381 0502
fax:                +358-(0)9-455 2103
mail:               Tietotie 2, Espoo
                       P.O. Box 1500
                       FIN-02044 VTT, Finland


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