![]() Then cameĬollege, and the revelation that the adults in my past were right allĪlong. Math teacher's quote meant nothing to me at the time. "To understand the universe is to understand math." My 8th grade But this could be one of your issues.ĥ.Use fresh reagents – grab new aliquots because you might be encountering contamination that is causing degradation.Īfter taking a look at our series of articles, you may not have to rely so much on the PCR good luck charms and superstition to get you through the task. It’s understandable that some larger rigs can go a few runs without being changed. Keep within 20-35 cycles.ģ.Reduce extension times / Raise annealing temperature – both of these will help improve your PCR results by reducing the occurrence of nonspecific binding and smearing bands.Ĥ.Change your TAE – For small gels, be sure you’re changing your TAE with every run. Try to reduce your template to see if that improves your results.Ģ.Lower cycle times – This is especially helpful when your template concentration is higher. Smeared Bands: There are several factors that might cause smearing to occur, and we have some simple solutions to fix that.ġ.Reduce your template – Having too much template seems to be the most common issue. when using T7 endonuclease I to digest negative controls. It might be a good idea to use fresh aliquots of your PCR material. annealed PCR fragments with T7 endonuclease I, if two shorter bands of the predicted size are. ![]() You may want to check the DNA quality and re-isolate if necessary.ĥ.Use fresh reagents – contamination is often an issue. Your concentration might be too low and increasing it could greatly improve your results.Ģ.Increase your cycle times – this is especially helpful if you’re still facing low template concentrations.ģ.DNA degradation could also be the issue. ![]() If you need to know exactly what is going wrong with your PCR, then it’s best to take it one step at a time.ġ.Check your DNA template. Some researchers will redo their PCR and have everything adjusted at once, while others will tweak one thing at a time to solve the problem. Weak Results: When encountering weak results, there are a lot of simple troubleshooting techniques to explore. In this article, I’ll go over some tips to help you when you’re encountering weak results and smearing. In the last two articles, I discussed nonspecific binding and what happens when you’re not getting any bands.
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