Fin DNA

J. Moore jlm37 at psu.edu
Tue Feb 12 17:41:33 EST 2002


Hi, 

Lately we have had a problem with DNA prepared from zebrafish 
tail fin clippings going bad. We have been doing a very simple 
procedure of incubating a small tail fin clip in lysis buffer + 
Proteinase K for 24-48 hours at 55 deg. C. A small amount of this 
is diluted in water, heated to 95 deg. for 10 min. to inactivate the 
Proteinase, and then used for PCR. Both the diluted DNA and the 
stock + Proteinase K is stored frozen at -20 deg.  What we have 
been seeing is that we get good PCR results from recently 
prepared DNA, but after a few weeks or a month or so, both the 
dilution and the stock sample won't amplify reliably. I'm not sure 
whether this is due to degradation of the DNA, or inhibition of the 
PCR amplification. And sometimes a piece of fin that has been 
stored in the freezer in lysis buffer prior to proteinase digestion 
won't provide PCRable DNA at all. Does anyone know what is 
going wrong or have ideas on how to prevent it?  

Thanks in advance,
Jessica

Jessica L. Moore, Ph.D.
Senior Postdoctoral Fellow
Jake Gittlen Cancer Institute, H059
Penn State College of Medicine
500 University Drive
Hershey, PA 17033

Lab: (717) 531-4704
Fax: (717) 531-5634
jmoore at psu.edu





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