proteinase K

Robert M. Mihalek mihalek at FORMULA1.smtp.anes.upmc.edu
Thu Mar 23 11:49:43 EST 2000


In article <v04011702b4fecf577bd4@[152.3.164.27]>
andreap at neuro.duke.edu ) (Portbury, Andrea (Chikaraishi Lab) writes:

> Hi,
> has anyone ever heard of having proteinase K activity problems after it has
> been stored in glycerol? We always used to store our stocks in either
> buffer or water, but this last time used glycerol. Since then, we have not
> been able to spool DNA purified from mouse tail cuts (i.e. to see the DNA
> we have to spin for 5 minutes) and our PCRs have not been working well.
> When we went back and used some old stuff that was prepared in water,
> everything was fine. The proteinase K in the glycerol appears to chew up
> everything o.k. - we get total disintergration of our tissue, but the DNA
> just seems to be either really dilute or not there at all.
> 
> Any help would be greatly appreciated

We use either water or a glycerol buffer to resuspend our proteinase K,
and do not see any difference in the quality of "spoolable" DNA from
our tail genomic DNA preps. I use Boehringer Mannheim PK along with the
following buffer:10mM Tris HCl, pH 7.5, 20mM CaCl2, and 50% glycerol,
and store at -20C.  I'll digest a 1 cm piece of tail in 400 tail lysis
buffer and use 10 uL of a 10mg/mL PK solution. Usually, I'll do the
tail digests for 2 hours at 55C in a rotary hybridization oven.


Bob Mihalek=================================================
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