Bcl digestion

[X. Y. Zheng]

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.
> 
> Hua

Hi, there:

  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.

Good luck.

X. Y. Zheng, Ph.D.