EcoRI problem

Eric Anderson e-anderson at ski.mskcc.org
Fri Jul 11 07:59:47 EST 1997


In article <33C50950.1059 at uvic.ca>, "Kris W." <kwilde at uvic.ca> wrote:

>The other day I was digesting a PCR-Script vector with EcoRI and I found
>that it cut at two sites when I was only expecting it to cut at one.  I
>normally leave the digestion for an hour or so but this time I left it
>for 3 hours.  I re-did the digestion for a half hour and found that
>there was only a single cut.  After looking at the sequence and the
>sizes of the fractions caused by the digestion I concluded that EcoRI
>(gaattc) cuts at aaattc when left for too long.  Similar sites such as
>gaattg were not cut.  
>
>Has anyone else noticed this or similar incidences with other
>restriction enzymes.

this is called star activity and is nicely reviewed on p. 211 in the back
of the NEB catalog, at least the 1995 version (an amazing reference for
nucleic acid/enzyme problems and questions).  they mention a 1975 PNAS
paper by Polisky et al. where they determined that "under conditions of
elevated pH and low ionic strength, EcoRI cleaves the sequence N/AATTN" 
they also mention that EcoRI is very sensitive to high glycerol
concentrations.  so if you used too much enzyme in too small of a volume
that might account for it.  other problems would be accidentally using the
wrong buffer (done that one myself more than once) or perhaps having
leftover EtOH in your DNA after a precipitation.  there is no mention of
long digests being a problem, but you never know, maybe nobody ever
checked (although i've personally never noticed that problem).

hope this helps,

eric

-- 
Eric C. Anderson
Sloan-Kettering Institute for Cancer Research 
Dept. of Cell Biology and Genetics
Lab: (212) 639-2977
Fax: (212) 717-3298
e-anderson at ski.mskcc.org



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