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
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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