EcoRI problem

brett brett at BORCIM.WUSTL.EDU
Fri Jul 11 08:31:25 EST 1997

>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.

Omigod. You've rediscovered star activity. To avoid this, try using much less
enzyme and use the appropriate buffer conditions. For more info, check out
a discussion of this problem in any decent molecular biology catalog, such as
NEB's. Best of luck.

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