Limited digestion

Daniel Auerbach auerbach at cell.biol.ethz.ch
Wed Feb 12 12:36:45 EST 1997


Hi everyone,

I'm trying to do a limited digest with Nsi I on a 5.5 kb plasmid. I first
cut with Eco 47 III and then add Nsi I. The aim would be that Nsi I cuts
only once, so that I can isolate the corresponding band. Unfortunately,
with every condition I try, Nsi seems to cut several times immediately (I
have 3 Nsi sites in the plasmid) resulting in a smear of bands and nothing
for me to isolate!

Can anybody tell me if there are conditions which influence an enzyme to
cut only once ? I tried low amounts, wrong buffer and lower temperatures,
nothing seems to work.

Thanks for any comments,

- Dan


-------------------------------------------------
*  Daniel Auerbach
*
*  Institute for Cell Biology
*  ETH Hoenggerberg
*  8093 Zuerich, Switzerland
*
*  phone: ++41 1 633 33 93, FAX: ++41 1 633 10 69
*  email: auerbach at cell.biol.ethz.ch
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