The non-specific bands could be from contamination of one of your stocks with foreign DNA (probably yours!). Re-do the reaction with a negative control (no template). Redesign the primers – try to stick to the guidelines as closely as possible. I find that DMSO is especially useful in problematic amplifications.ġ2. Try the reaction in another cycler – the calibration of the one you are using may be off.ġ1. Check the cycler – are the temperatures and times as you expect?ġ0. Try 5-10 parallel reactions with concentrations from 10 to 200 ng in a 50 microlitre reaction.ĩ. Your template concentration may be too low or the concentration of impurities in your prep may be too high. Try reactions with varying template concentrations. The best way to find the optimum temperature is to use a gradient cycler and test a range from the lowest primer Tm to 10✬ below in 1✬ increments.Ĩ. On the other hand, an annealing temperature that is too low can result in such non-specific priming that don’t allow specific bands to arise. If the annealing temperature is too high, you obviously won’t get any priming at your desired sequence. Otherwise, re-do the PCR with a mixture of Taq and 1/10th concentration of your proof-reading enzyme and you should still get the same amplification with a lower error rate.ħ. Remember to sequence the insert though – if you are lucky there won’t be any significant mutations and you can use the insert as it is. If an amplification is problematic with a proofreading polymerase, I often try using good old Taq and this often solves the problem. Re-make the template DNA, especially if you are working with genomic DNA. dNTPs can be destroyed by repeated freeze-thaw cycles.ĥ. Check that the primers have been diluted to the correct concentration.Ĥ. Check that the polymerase buffer has been fully thawed and mixed thoroughly.ģ. Try the reaction again, you may have left something out.Ģ. I will add your ideas to the list to make it a resource we can all refer to. I’ve inevitably missed some things out, so please chip in if you can think of anything else to add. Today I have brainstormed all of the ways I can think of to approach problems with standard PCR reactions. Even with the simplest PCR reaction things can go wrong, so you need to have a good checklist of ideas for PCR troubleshooting and rectifying the problem. Try to check your PCR product by electrophoresis or by melting analysis.Routine PCR? Let’s be honest, there’s no such thing. It is possible that your primers and probes generate dimers and your PCR gives you false positive signal in your analysis. There is a problem with primers and probes design.Please repeat the analysis to exclude this problem. There is a possibility that you have added the template into your NTC reaction.If there is positive signal in negative control (or no template control), there could be some of the following issues: Be sure to have adequate time and temperature for all steps of your PCR. There is an issue with thermal profile optimization.It is also possible that the probe is not designed properly and doesn’t bind to the amplified product. The probes synthetized in Generi Biotech may have up to 50 freeze-thaw cycles. The probe could be partially degraded because of high amount of freeze-thaw cycles or wrong long-term storage. There is problem with Probe quality or design.Next option is usage of internal positive control that can check the correctness of PCR. The solution: It is appropriate to use additives (BSA, DMSO, Betaine, etc.) to enhance your PCR. It is possible that your template contains inhibitors that cause poor PCR, for example salts, SDS, phenol, plant and human tissues or fluids. This can sometimes help you to save time and nerves in solving the problem. It looks like a trivial cause of no signal in your PCR, but always check again that you have added the template to your PCR. If there is no signal in your real-time PCR results, there could be some of the following issues: We will try to help users with the main reasons of your PCR troubles and how to solve them. Their real-time PCR curves give no signal or they have complication with signal in negative control. PCR users have sometimes trouble with their real-time PCR experiments.
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