RT-PCR, DNA contaminant removal by digestion with restriction enzyme - (Mar/09/2006 )
Instead of a DNAse I digestion is it possible to do some digestion by restriction endonuclease that cut the DNA between the two primers for amplification by pcr ?
What do you think abotu that ?
I think it can work if the contaminant is low yield. I'll do some test when I have the chance.
-Christian Croisetière-
Hi,
I was using successfully DpnI restriction enzyme. It cuts methylated DNA and beautifully removes DNA contamination in RT-PCR reactions.
Good Luck!
Ilona
-ili_-
QUOTE (Christian Croisetière @ Mar 9 2006, 02:17 PM)
Instead of a DNAse I digestion is it possible to do some digestion by restriction endonuclease that cut the DNA between the two primers for amplification by pcr ?
What do you think abotu that ?
I think it can work if the contaminant is low yield. I'll do some test when I have the chance.
What do you think abotu that ?
I think it can work if the contaminant is low yield. I'll do some test when I have the chance.
PCR is too sensitive. Even if the RE misses just a few molecules, you might see the signal.
I would suggest Shrimp nuclease, which works pretty much like DNaseI, but has higher activity (need to add less), and is very easily heat inactivated.
-Gerd-
Actually there was a paper on exactly this in Biotechniques recently.
Simple enzymatic means to neutralize DNA contamination in nucleic acid amplification
John Ashkenas, James W. Dennis, and Chi Yip Ho
BioTechniques Vol. 39, No. 1: pp 69-73 (July 2005)
Works very well, apparently
-John Buckels-