X. Y. Zheng
xi-zheng at tamu.edu
Thu Feb 26 11:59:10 EST 1998
Xiao-Hua Xue wrote:
> Hi, dear netters,
> I used BclI (from GIBCO) to digest a plasmid and got some problems: 1)
> there is only very weak band showing up which is supposed to be the
> digested DNA, while undigested DNA still show high density. I assume this
> is due to incomplete digestion. 2) there is a big smear in front of that
> weak digested DNA band. I assume this is due to nonspecific digestion. I
> tried a few times by changing concentration of BclI, digestion time (from
> 2hr to overnight), at temperature 50oC and results are similar.
> Does anybody have experience on BclI digestion. I really appreciate if
> you could give me some suggestions. Thanks in advance.
Be sure this enzyme is methylation sensitive, i.e. its activity will
be blocked by dam methylation. To solve the problem use specific E.
coli strain that does not contain three site-specific DNA methylases.
X. Y. Zheng, Ph.D.
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