Degenerate primers PCR problem, Please help!
Posted 26 October 2010 - 09:01 AM
I am planing to amplify gene cassette from samples using the degenerate primers pair HS286 and HS287 published by Stokes et al. in 2001 (Appl Environ Microbiol. 2001 Nov;67(11):5240-6). In fact, I am having difficulties using these primers in my PCR since I used them several times without a reproducible results. I ordered these primers from Invitrogen five times (two of which were HPLC purified). Typically, the PCR gives the ideal bands (from 300-1,500 bp) described by stokies for only a couple of PCR runs. Then, I notice there is an increase in primer dimer and decrease in products. Eventually, the PCR fail to work using exactly same conditions and sample, and the only product that I get is a strong primers dimer when analyzed on agarose gel.
I extracted the DNA using FastDNA SPIN kit (MP Biomedical). Quality of my DNA is good, and amplification using other PCR primers and this extracted DNA works fine.
I tried both regular PCR and hot start PCR protocol. Also, I tried gradient PCR and touch down PCR protocols, but still no luck.
I tried 0.1, 0.25, and 0.5 UM of each primers. I also tried, 5, 10, 25, and 50 ng of template DNA in 50 and 100 ul PCR reactions.
I optimized the Mg concentration from 0.5-5 mM, increasing the concentration 0.5 mM in each reaction tube.
I tried Taq DNA Polymerase (Invitrogen cat No. 18038-240), Platinum® Pfx DNA Polymerase (Invitrogen Cat No. 11708-013), iScript™ One-Step RT-PCR Kit With SYBR® Green (Bio-Rad Cat No. 170-8892) , Phire® Hot Start II DNA Polymerase (Finnzymes Cat No. F-122S), and Phusion™ Hot Start High-Fidelity DNA Polymerase (Finnzymes Cat No. F-540S) with and without DMSO with the last enzyme only. Still no luck.
I also, ruled-out human error and equipment error by sending my DNA and primers to other people in other labs, and PCR failed too.
At that time, I was thinking may be it is the primers synthesis or chemistry. So, I ordered a new pair from Sigma-Aldrich Co. Unfortunately, it did not work either.
So, is there other hints that I should keep in mind in order to make my PCR work?
I really appreciate your help,
Posted 27 October 2010 - 12:35 AM
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Posted 27 October 2010 - 05:49 AM
I did. Still no reply
What is weird is that other people has used the same strategy and the same primers without any problem.