I've been having trouble with a PCR reaction. I ran two PCRs with the same DNA samples, the only different being the primers. The first PCR (labeled Zip14 WT) worked fine. The second PCR (labeled Zip14 KO) showed a smear in every lane. I was using brand new primers from IDT. Any thoughts?
Dear Aaron,
Smirred PCR products are usually due to a cDNA or mRNA degraded templates. Did you assess the quality of your RNA before synthesizing your cDNA? Did you your WT and KO cDNA with the same annealing temperature?
Have you optimized your PCR mix reaction and PCR condition? Maybe higher annealing temperature or using different PCR methods such as touch down PCR.
You can also recheck your primer's quality. Are they specific, etc? If none worked, maybe you should redesign your primer.
New primers look non-specific (at least one of them). If you optimize the PCR, you see a few major bands. Re-design primers, and test at least two-three pairs.
You can use gradient PCR to find out the exact annealing temperature of your new primers.
This problem also can be due to breaking down the primers because of intense pipetting of your primer stock or master mix. Also you should make sure that all your reagents and microtubes are DNase free.
you can see whether the DNA extracted in right way and also check the gel electrophoresis because sometimes due to temperature and volt set in the electrophoresis shows smear in PCR.
Sometimes using less of the DNA sample in the PCR reaction reduces the number of amplicons caused by non-specific binding and removes the smears. It worked for us and the "smeared PCR", after the trouble shooting, became a reliable diagnostic PCR!
I agree with the above suggestions that you might have slightly degraded RNA template or that the primer design can be the mistake. You might also have overloaded your gel, if you have a large yield. Try then to dilute you sample before applying it to the gel. A smear can also be due to overrun of the taq polymerase making the amplicons a little longer for each cycle. This can be overcome by using primers with a 5' modification like for example SuPrimers.
Dear
I think your second primers are non-specific and don have good quality. order the second primers from another company.
you say that the dna samples are the same but all of the samples that did not work are labelled KO. Is there something in the knockout process which has destroyed/modified one primer annealing site so that only one primer is working properly in which case you can expect smears particularly if the non-working primer was the more specific of the pair ( fewer blast hits against your genome ). do you get the same result if you run the pcr of the knockout samples with only one primer perhaps?
Hi All,
Thanks for your suggestions. To elaborate on what I am doing, this is a simple genotyping PCR using genomic DNA. The top PCR is for the wild type allele and the bottom PCR is for the knock-out allele. It's highly unlikely that the problem stems from degraded DNA. I've tested the same samples with multiple primers and have had success with every PCR but the one shown. Therefore, the problem is specific to this primer set. The first two times I ran this PCR I had success (there were no smears and the only product was the band expected), but it has since given me the shown results every time. We even tried ordering new primers, with no success.
Another researcher at my institution suggested adding DMSO to the reaction, which I will do next. I will also try running a gradient for the annealing temperature, as suggested.
Thanks again for your input. It is highly appreciated!
Hi Aaron,
Did you get the above PCRs to work?
I am facing the exact same problem that you are facing. I am carrying out genotyping with samples from Arabidopsis. I have checked the genomic DNA with other primers, and the PCRs worked. I had carried out gradient PCRs to stndardize the conditions. And had determined conditions suitable for carrying out PCRs. However, I am unable to repeat them, and I see smearing like the one that you observed. I have changed all the reagents, tried using different amounts of template, have varied DMSO concentration, ordered new primers, used Taq, phusion polymerasaes.
Any inputs would be highly appreciated.
Thanks,
Indu
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