Dr. Duncan Clark
duncan at genesys.demon.co.uk
Thu Feb 26 11:23:32 EST 1998
In article <199802252230.OAA24886 at fraser.sfu.ca>, Xiao-Hua Xue
<xxuea at sfu.ca> writes
>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.
Bcl I (TGATCA) will only digest dam- DNA (dam recognises GATC and
methylates the A). You will need to prepare your plasmid from a dam-
E.coli mutant ie JM110, GM48, GM2929, GM2163 or SCS110 spring to mind.
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
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