For the first 10 months of me being in my permanent lab, our genotyping PCR was more or less a breeze, and worked wonderfully. However, it started working only about half of the time in October, and now it will not work at all. I've included an image of a gel I ran today. Sample 403 is a known positive control that used to work but no longer does. The four samples in the middle are new ones with unknown genotypes, and the three on the right are aliquots from nuclease-free water vials. I ordered new primers, prepared new solutions of MgCl2 and betaine (Q solution), and used new nuclease-free water. I'm running out of options, and my lab is now super backlogged, with around 100 mice that don't have genotypes. Please, if anyone has found a solution to a similar problem, let me know. The most puzzling part is that used to work all of the time, and now is a consistent failure.
Many thanks.
When water or no template controls give an obvious amplification then you have post pcr contamination in a common reagent, in your working area (dust carried) or in your equipment. The quick and certain way of dealing with contamination is to design one or both primers outside of the current amplimer then the contamination cannot amplify and you just get a slightly larger product. Failing that go to another lab or distant working area in your lab.Use their pipettes and pcr reagent and pcr machine and run all variations of your primers on a dna sample that has worked befor.eg oldF and newr, newf and old R, oldf and oldR and newF and newR with a no template control for each primer set ( better 2 NTCs) and see if you can isolate the contamination
Dear Jennifer
Having bands with water mean your mastermix/ PCR mix has carryover contamination. Solutions available at Article Setting up a PCR laboratory
Dimers are present in all the samples loaded on the gel, so it means PCR reactions (polymerase) are working.
Think about DNA extraction, someone changed the concentration of something? Run your DNA samples on a gel to confirm if you really have DNA
Best
Mustafa Munawar
www.linkedin.com/in/mustafa-munawar
Dear Jennifer,
I also suspect that there is something wrong with your DNA template prep. I agree with Mustafa: first confirm if you have DNA at all (including in the positive control). Are you sure that there is no EDTA, ethanol, etc? Additionally, I would try to set up reactions without using master-mixes, but by adding all the components separately.
Best wishes
Anton
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