Mutagenesis Kit

Wolfgang Schechinger wolfsc at ibms.sinica.edu.tw
Sun Sep 9 21:47:04 EST 2001


Dear Amna,

yea, a long DpnI step is important (and shouldn't harm your mutagenesis)

>  Do a long DpnI digestion - I digest for at least 2 -3 hrs

... and do not disturb (i.e. spin down) the liquid phase afterwards or perform 
things like phenol extraction in the same tube. Transferring the reaction 
carefully to a new tube before downstream processing might reduce 
background, too. 
There always seems to sit some undigested  vector somewhere on the wall 
or under the lid of your tube that you might re-introduce into your reaction 
mix when you spin it down. 
I noticed a drastically decreased (virtually no) background in ligations when 
using vector (double digest + insert killing enzyme, CIP, one tube rxn) after 
phenol extraction.

I'd appreciate if somebody would like to comment that observation!

Wo




[tiss mezzage wahs broduceRd using TYPO GENERATOR zoffwer]
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Dr. Wolfgang Schechinger
Lab N233 (c/o Dr. Steve Roffler)
Institute of Biomedical Sciences
Academia Sinica
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Taipei 115
Taiwan R.O.C.
Tel +886-2-2789-9152
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Mobile +886-925-136893
e mail wolfsc at ibms dot sinica dot edu dot tw
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