Bcl digestion

Hiranya Roychowdhury hroychow at NMSU.EDU
Thu Feb 26 12:50:42 EST 1998


At 02:31 PM 2/25/98 -0800, 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  
>
>

What host are you getting your plasmid from? BclI site is methylated in most
commonly used hosts. Try getting your plasmid from GM33 or a similar dcm-
strain.


Dr. Hiranya Sankar Roychowdhury
Plant Genetic Engineering Lab.
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroychow at nmsu.edu




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