I am working on PCR since last 12 years I never had problem with dNTPs. Recently I moved to new lab and started working on gene cloning. My old reagents for PCR were ended and I ordered new reagents. Taq Polymerase (Sigma- same as old one) is working fine, but when I ordered new set of dNTPs I had so many problems with it. I never got results with new dNTPs, I tried dNPTs from 3 different companies but non of them s working (From cheap to expensive) (GE, Thermofisher, Invitrogen etc.). Although my old dNPTs are working fine. I contacted every supplier and they sent me new set but still it is not working. I also tried so many combinations with new set with different primer concentration, different annealing temperatures (with gradient PCR) but non of them is working. I took some working dNTPs from other labs, they are working fine (they are from the same suppliers as I am using). I am in trouble now as my old dNTPs are going to end soon. Does anyone have such issue or any suggestion? Thanks
It sounds like some common problem that is confined to your lab, like perhaps there is a problem with the water or buffer you are using to dilute the dNTPs or you did the calculations wrong for dilutions. Otherwise I can't imagine why 3 different sets of dNTPs are not working.
Probable causes are:
1. The concentration of dNTPs may differ from your previous batch. Since the dNTP concentration and needed Mg++ concentrations in the PCR reaction are related, you might want to try titrating your Mg++ concentrations.
2. Probably your primers were contaminated, not the dNTPs.
3. Vortexing may inactivates the Taq polymerase and hinders the reaction.
Dear Hitesh, to me it also sounds like a problem that is not caused by the dNTPs. It might well be that you have one batch that is not O.K., but it is very unlikely that there are 3 different sets that do not work. Which water do you use? Self-distilled? I can recall an episode where we used water that was not properly distilled. Thus we did PCR actually using tap water... Some PCR reactions still worked, but the majority did not work at all. As you have tested 3 different sources of dNTPs you can almost rule out that they are the sole problem. I can imagine that salt concentrations could be the issue and would like to recommend that you use different water and also try a different buffer.
I hope you can solve the problem :-)
Best regards,
Moni
Thank you Michael J. Benedik , Bassam MS Al-Musawi and Monika Stefanie Schütz for you comments.
Regarding Michael's comment, one of my dNTP is ready to use so I did not mess with the concentration. Bassam MS Al-Musawi I changed my my all reagents to fresh stock, like primers, Taq, water, buffer. I also avoided vertexing, I need to try Mg++ concentrations.
Monika Stefanie Schütz I do understand that water could be the factor, I also used different water (commercial nuclease free water, autoclaved distilled water, RO water, water from different kits).
I have one more question for you, does the TE buffer inhibit PCR reaction? as I dissolve my DNA in TE buffer (that could be a factor).
Once we had similar problem. We got Taq polymerase from a new supplier, which worked only with the dNTP mix supplied with it. This Taq refused to work with any other working dNTPs (from different supplier) available in our lab.
SD
Yes Sibnarayan Datta , I do have old dNTPs from invitrogen which are working absolutely fine (about to end). I also ordered new Taq polymerase to check if current one is not compatible with new set of dNTPs. I tried dNTP mix from Sigma (Borrowed from other lab) which is working good. I am really curious to figure out what is the problem.
Thank you everyone for your comments and suggestions. I figured out, what is the problem. It's my DNA sample, which I dissolved in TE buffer. I cleaned up my DNA with DNA clean up kit and dissolved it only in Distilled water no buffer. After the PCR reaction I got bands in all the dNTPs that are not working previously.
Thank you !!
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