qPCR on genomic DNA - (Nov/03/2004 )
I have been trying to quantitate a region on the DNA from humans for the past few months. First, realized the primers which the Primer2 Express (ABI) gave me were crap and hence redesigned them with Primer3. A base separates the 3' end of Forward primer from the 5' end of the TaqMan Probe. However, the 3' end of reverse primer is 39bp away from the 3' end of the probe. The region I am trying to amplify can be amplified by a cold PCR using the above primers and the sequence of the region is also intact. Futile attempts so far conducted,
1) At 60 C, i get good amplification from the BAC DNA, but weak from genomic normal DNA
2) Changed the annealing temp from 60 to 62 C-nothing!
3) Used 2X probe-again no big difference!
4)Increased the time for denaturation from 15s to 30s-samples evaporated!
So close (to graduating), still so far away!! Need this last bit of data!
Any suggestions on qPCR on DNA?
Thank you all,
are you shure of the sequence (any SNPs)? Have you tested the quality of the DNA, is it pure, and the correct concentration (too high or to low can inhibit the reaction).
Try a diluted sample of the DNA to test for contaminants in the purified DNA.
Søren M. Echwald, MSc., Ph.D.
One Real-time PCR kit, which covers 38.565 genes
Thanks for your suggestion. My PCR is 'kind of working'. I doubled the amount of the primer that was further away from the probe. I get amplification from around 26-27 cycles using 20 ng of genomic DNA. More DNA did not give me any significant difference in the amplification plot. I am bit concerned though about an 'arc' that shows up late (36-38 th cycle) in the non-template (NTC). Coud this be due to some primer-primer/primer -probe combination incompatibility (OR DNA contamination !!)? NTC is clean otherwise. Your suggestions appreciated.
Forgot to add, I had tested four different DNA (Normal) for possible SNPs when nothing was working! All intact!