When doing PCR, you can encounter all sorts of issues, which is why we have been doing a series on troubleshooting tips for different obstacles that occur. In the last two articles, I discussed nonspecific binding and what happens when you’re not getting any bands. In this article, I’ll go over some tips to help you when you’re encountering weak results and smearing.
Weak Results: When encountering weak results, there are a lot of simple troubleshooting techniques to explore. 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. 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.
1.Check your DNA template. Your concentration might be too low and increasing it could greatly improve your results.
2.Increase your cycle times – this is especially helpful if you’re still facing low template concentrations.
3.DNA degradation could also be the issue. You may want to check the DNA quality and re-isolate if necessary.
4.Increase your primers.
5.Use fresh reagents – contamination is often an issue. It might be a good idea to use fresh aliquots of your PCR material.
Smeared Bands: There are several factors that might cause smearing to occur, and we have some simple solutions to fix that.
1.Reduce your template – Having too much template seems to be the most common issue. Try to reduce your template to see if that improves your results.
2.Lower cycle times – This is especially helpful when your template concentration is higher. Keep within 20-35 cycles.
3.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.
4.Change your TAE – For small gels, be sure you’re changing your TAE with every run. It’s understandable that some larger rigs can go a few runs without being changed. But this could be one of your issues.
5.Use fresh reagents – grab new aliquots because you might be encountering contamination that is causing degradation.
After 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.
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