PCR's which produce DNA concatermers

jsowden at uwa.edu.au jsowden at uwa.edu.au
Tue Jul 30 11:56:53 EST 1996


I have been successfully amplifying marine snail mtDNA fragments, using 
PCR, until recently. My problem is the appearance of very long DNA 
fragments (concatermers) which stick in the well when I analyse the PCR 
product by agarose electrophoresis. The mucopolysaccharides in the 
snail tissue can interfere with the PCR and I am currently attempting to 
ensure that my mtDNA template is clean.

I have confirmed that the concatermer does consist of the targeted PCR 
fragment, as follows. I had, previous to this problem, carried out 
restriction analysis of the DNA fragment and was able to retrieve the 
correct sized DNA fragments by an overnight digest of the concatermer 
reaction. 

I am setting up new reagents for the PCR and making up fresh primer stock 
dilutions from my original concentrated stock which has been stored at -20C. 

What is causing the amplified fragments to concatermerise and 
how can I prevent it? Are my primers failing or is the polymerase the 
most likely suspect? 

Any light that can be thrown will be appreciated.

TIA

jan.
---------------------------------------------------
[ Posted via Deja News:  http://www.dejanews.com/ ]



More information about the Methods mailing list