caulfield_z at yahoo.com
Tue Oct 1 09:23:18 EST 2002
I have ligated vector+insert and transformed by electroporation into EColi.
I obtained colonies and I screened them by PCR (I take a bit of the colony and
I lyse it and perform a PCR specific for the insert on it). I seem to have
obtained the correct colonies. When I do a plasmid extraction by miniprep
(Quiagen) I obtain a plasmid band. But the problem is when I try to digest
the plasmid with restriction enzymes (tried several) I obtain a smear on gel!
A control of plasmid extracted in RE buffer does not induce the degradation
therefore I assume it's not caused by DNAses. The parent plasmid is well
digested however. What could be going wrong? It's not possible that RE smear a
plasmid that originally would be cut by them only 2 or 3 cuts!
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