Problems with SacI
Lemee at ibm.net
Fri Jun 11 09:49:35 EST 1999
Sounds to me that either the RE is near dead or the DNA is dirty (or not
what you think it is). Regarding the double digest, if you dump too much
REs, then you get too much glycerol in the final volume which is not good
(most RE are in 50% glycerol, or something like that, 5% final is nearing
the upper limit). SacI is a common RE, it should work.
Hope you get the situation resolved.
Martin Offterdinger wrote:
> I am currently facing serious problems using the SacI restriction
> enzyme. I can cut a purified plasmid with it, but I am not able to do
> a double digest within the same buffer. Nor can I do the digests
> sequentially, i.e. digest with second enzyme first, gel purify
> fragment, redigest with Sac I. So what else could I do? I have heard
> that Sac I is extremely sensitive to salt, so should I try to wash the
> fragment more extensively ??
> Martin Offterdinger
> Internal Med.I,Dept. Oncology
> University of Vienna
> E-Mail:a8803349.nospam at unet.univie.ac.at
> (remove .nospam before mailing)
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