BamHI Without Star Activity

Paul N Hengen pnh at fcsparc6.ncifcrf.gov
Thu May 19 15:39:11 EST 1994


 In article <smrodems-180594221130 at f180-206.net.wisc.edu>
 smrodems at students.wisc.edu (Steve Rodems) writes:

| I am having some trouble getting a clean BamHI digest of a plasmid without any
| extra bands.  I am using 5X excess of enzyme by units, and am using a standard
| buffer with 150 mM NaCl.  It is important that I get only linear plasmid, and I
| can't gel purify the fragment for other reasons I won't get into now.

> My first suggestion would be to use BamHI from another company than the one
> your using.  I have never had problems with BamHI from BRL.  This enzyme
> cuts in REact 3 which is 100mM NaCl.  Maybe you buffer is too high in salt.
> I have never used BamHI from NEB but several other enzymes from NEB work
> well in my hands so they may be a good source as well.

Assuming that the extra bands are from non-BamHI sites recognized, ie. star
activity and not from incomplete partial digestion, you might want to tinker
with the recognition specificity of the enzyme by adding some spermidine to the
DNA sample before the restriction digest.

@article{Pingoud1985,
author = "A. Pingoud",
title = "Spermidine increases the accuracy of type {II}
restriction endonucleases.
Suppression of cleavage at degenerate, non-symmetrical sites.",
journal = "Eur. J. Biochem",
volume = "147",
pages = "105-109",
year = "1985"}

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* Paul N. Hengen, Ph.D.                           /--------------------------/*
* National Cancer Institute                       |Internet: pnh at ncifcrf.gov |*
* Laboratory of Mathematical Biology              |   Phone: (301) 846-5581  |*
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