Bcl digestion

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....

Duncan Clark
DNAmp Ltd.
TEl/FAX 01252376288

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