4 replies to this topic

[jessh]

Hi all!

I am using Deep Vent polymerase to amplify a region in a GOI in genomic DNA. I initally optimised my reaction conditions on plasmid DNA containing my GOI, but when I tried to amplify my GOI in genomic DNA I got no product.

The plasmid DNA and genomic DNA templates were diluted such that they have equal amounts of GOI target i.e:
1.07 pg pDNA = 160, 000 copies
and 1 ug gDNA = 160, 000 copies

The plasmid alone amplifies well, however when spiked into null genomic DNA I get no product! What is it about the background of genomic DNA that could be causing inhibition for Deep Vent??? I have tried this reaction with regular taq and it works perfectly. Could the exonuclease activity of Deep Vent have something to do with it?

Any suggestions or comments would be greatly appreciated!
Thanks
Jessica

Edited by jessh, 10 February 2010 - 08:21 PM.

[phage434]

PCR inhibitors in your gDNA sound like the problem.   Try a 10x or 100x dilution of your template DNA and a few more cycles.

[jessh]

phage434, on Feb 11 2010, 03:41 PM, said:

PCR inhibitors in your gDNA sound like the problem.   Try a 10x or 100x dilution of your template DNA and a few more cycles.

Thank you for your reply.
I have tried 100 ng genomic DNA, however this still did not work. For my application I need to ideally amplify from 1 ug DNA and I really couldn't go any lower than 100 ng. My genomic DNA is in dH2O - what kind of inhibitors do you think it could be? I use quite a good kit to extract my gDNA.

[phage434]

I don't understand where your requirement of a minimum amount of template comes from.  You need only a single molecule, in principle.  Have you tried lowering the annealing temperature and lengthening the extension time and increasing the number of cycles?  Could you tell us in detail your reaction composition and cycling conditions?  I would recommend TE for dissolving your gDNA rather than water, to control pH and reduce Mg++ DNAse cofactors.

[jessh]

phage434, on Feb 12 2010, 12:22 AM, said:

I don't understand where your requirement of a minimum amount of template comes from.  You need only a single molecule, in principle.  Have you tried lowering the annealing temperature and lengthening the extension time and increasing the number of cycles?  Could you tell us in detail your reaction composition and cycling conditions?  I would recommend TE for dissolving your gDNA rather than water, to control pH and reduce Mg++ DNAse cofactors.

I ultimately want to amplify from gDNA that has been isolated from cells that have been highly transduced with a viral vector. This will create a complex library of unique site, hence the more DNA, the more unique integration sites I will be able to detect.

I have solved this problem by using a different enzyme (Pfu native) and adding DMSO (1% only). The deep vent didn't work when I used betaine OR DMSO. The Pfu didn't work with betaine (5M) or higher amounts of DMSO (5%). I do have reduced yields when using the larger amounts of gDNA (1 ug) but the reaction does seem to work well with 800 ng. One other thing I did try which seemed to help was using a 100 ul reaction volume, instead of 50 ul.