Plasmid construction

WS via methods%40net.bio.net (by novalidaddress from nurfuerspam.de)
Tue May 8 11:16:43 EST 2007


Dear Yvonne,

as you have two incompatible RE sites, you may do some quick and dirty
molecular biology as follows:

dephosphorylate your insert (you may add some CIP or SAP to your
digest), then run a PCR cleanup (or make a phenol/chloroform
extraction)

additionally, when you cut out your insert, add one or two more
enzymes that cut outside your insert and make pieces of vector 1. In
the same way, you might add another enzyme or two that cut inside the
region you need to cut out from pCDNA3 and then do a cleanup as above.

Both additional cuts will create additional fragments that cannot
directly ligate into your opened pCDNA3 and thus are much less favored
ligations (as you need to ligate 3 or 4 fragments). Thus most of your
clones will carry the desired insert. Easy to check by digesting quick
and dirty minipreps done by the alkaline lysis protocol.

HTH

Wo



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