Inactivation of Proteinase K

Hiranya Roychowdhury hroychow at NMSU.EDU
Wed Oct 30 12:35:10 EST 1996


At 06:38 PM 10/30/96 +0100, Masato Furuichi wrote:
>Hi,
>
>We use high concentration (2mg/ml) of proteinase K and then ethanol 
>precipitation to purifiy DNA from cellular extracts. After that some 
>enzymes doesn't work efficiently, probably due to the rest of 
>proteinase K in the precipitate, I need help for a protocol in order 
>to inactivate proteinase K efficiently, other than phenol extraction.
>ijiwaru at wheel.dcn.davis.ca.us wrote:
>> 
>
>Thanks
>
>Agnes.Cordonnier at esbs.u-strasbg.fr (Agnes Cordonnier (UPR9003-CNRS))
>
>

I am not sure if you could remove all the protein from the DNA sample
without PCI extraction. However, proteinase K may be inactivated by heating
the sample at 75 C for 10min in the presence of 10mM EDTA and 0.01% SDS.
Following that, the DNA should be pptd with ethanol using ammonium acetate a
few times to get rid of as much SDS as possible. One could also try to
purify the DNA on a DEAE column but for genomic DNA this is tricky.


Dr. Hiranya Sankar Roychowdhury
Plant Genetic Engineering Lab.
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroychow at nmsu.edu




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