PCR (Polymerase Chain Reaction), which is so common to the lab, can be very frustrating. We understand that for researchers, especially those who are just starting out, PCR can be a nightmare. So we’re breaking down PCR troubleshooting tips into a series of articles.

So what do you do if you're running a PCR or series of PCRs and suddenly you're not getting any bands?

There are several troubleshooting tips when you don't get PCR bands, but some basic steps is to ensure you have an organized master mix, your are confident about your PCR concentrations, and you aliquot everything. But there are other important tips to help.

 

In the last article, I discussed what to do when you get nonspecific binding. And in this article, I’ll provide some tips to help when you encounter a PCR yielding no results. For veteran researchers, we’d like to open this up for discussion. Provide your own tips in the comment section. The shared information can be so useful for others.


1. Organize your Master Mix:

The first thing is to make sure you didn’t forget anything. Get in the habit of maintaining a certain order when making your master mix. It can also really help to use a checklist. Not only is it a reminder of what to add, but it will also jog your memory when checking back over your work. After imaging your gel and seeing you results, you now have that checklist as a visual cue. You’ll be able to think to yourself upon seeing it, “Yes, I remember adding everything, so that can’t be the problem.” Or, “Oh no! I forgot to add the taq.” On your checklist you should have: water, buffer, dNTP, MgCl2 if it’s not in your buffer already, primers, taq polymerase and your template.

2. Check Concentrations:

The key to PCR is keeping everything optimized. Having too much of one thing can cause inhibitory effects. Sometimes it’s just a matter of needing to dilute your template, or reducing your dNTP.

3. Aliquot Aliquot Aliquot:

This is a tip from the last article, and it’s worth repeating. Freeze –thaws can really spoil your reagents and your template. Remember, it’s not just about pulling out stock and putting it back into the freezer – the location in an upright freezer can also impact its shelf life which was discussed in an earlier article about fridge/freezer organization. This tip is really important for ensuring your PCR goes smoothly. It’s a tip I wished I had been taught earlier in my lab experience.

4. Increase your PCR Cycles:

This is especially important when your template concentration is very low. Bumping up the number of cycles could get you exactly where you need to be.

5. Consider PCR Additives:

There are a wide variety of PCR additives that help enhance the process, including BSA and DMSO. BSA helps prevent your PCR components from sticking to the vial. The suggested use is up to 0.8 mg/ml. DMSO is especially helpful when dealing with high GC templates because it greatly relieves the secondary structures. It’s best to use it at a concentration of 5%-10%.

Special Circumstances:

What if the problem is that certain bands are showing up, but other bands which are expected to show up are not coming through? It’s these special situations that can really drive a person crazy during PCR. So let’s go through a few options to help when this happens.

1. Dilute Your Template:

This goes back to checking your concentrations. And it happens quite often that your template just needs to be diluted to cause fewer problems.

2. New Primers:

If you’re working with an old stock of primers, it’s possible that you’re encountering some degradation. Try working with fresh stock and then run your PCR again. And when working with your new aliquots, be sure they are fully thawed.

3. Increase Your MgCl2:

Remember magnesium chloride helps to enhance extension and increases the activity of taq. Increasing your MgCl2 can really boost your PCR results. But with that said, it’s important to do this carefully because it can reduce specificity.

4. Annealing Temperature:

If you’re coming across strong dimerization, it might help to increase your annealing temperature. Or even try touch-down PCR (which is explained in the previous PCR article).

If you have more suggestions, please comment below. Keeping a library of troubleshooting hints is beneficial to every researcher.

Continue following this series for more PCR tips. In the upcoming articles, I’ll explain what to do when encountering smearing, weak results and contamination.


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