![]() ![]() Then cameĬollege, and the revelation that the adults in my past were right allĪlong.Causes Related to Cycling Times and Temperatures Math teacher's quote meant nothing to me at the time. "To understand the universe is to understand math." My 8th grade In the upcoming articles, I’ll explain what to do when encountering smearing, weak results and contamination. Keeping a library of troubleshooting hints is beneficial to every researcher.Ĭontinue following this series for more PCR tips. If you have more suggestions, please comment below. ![]() Or even try touch-down PCR (which is explained in the previous PCR article). But with that said, it’s important to do this carefully because it can reduce specificity.Īnnealing Temperature: If you’re coming across strong dimerization, it might help to increase your annealing temperature. Increasing your MgCl 2 can really boost your PCR results. Increase Your MgCl 2: Remember magnesium chloride helps to enhance extension and increases the activity of taq. And when working with your new aliquots, be sure they are fully thawed. Try working with fresh stock and then run your PCR again. New Primers: If you’re working with an old stock of primers, it’s possible that you’re encountering some degradation. And it happens quite often that your template just needs to be diluted to cause fewer problems. So let’s go through a few options to help when this happens.ĭilute Your Template: This goes back to checking your concentrations. 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. It’s best to use it at a concentration of 5%-10%. DMSO is especially helpful when dealing with high GC templates because it greatly relieves the secondary structures. BSA helps prevent your PCR components from sticking to the vial. PCR Additives: There are a wide variety of PCR additives that help enhance the process, including BSA and DMSO. Bumping up the number of cycles could get you exactly where you need to be.ĥ. PCR Cycles: This is especially important when your template concentration is very low. It’s a tip I wished I had been taught earlier in my lab experience. This tip is really important for ensuring your PCR goes smoothly. 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. Freeze –thaws can really spoil your reagents and your template. Sometimes it’s just a matter of needing to dilute your template, or reducing your dNTP.Īliquot Aliquot Aliquot: This is a tip from the last article, and it’s worth repeating. Having too much of one thing can cause inhibitory effects. 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, MgCl 2 if it’s not in your buffer already, primers, taq polymerase and your template.Ĭheck Concentrations: The key to PCR is keeping everything optimized. After imaging your gel and seeing you results, you now have that checklist as a visual cue. Not only is it a reminder of what to add, but it will also jog your memory when checking back over your work. It can also really help to use a checklist. Get in the habit of maintaining a certain order when making your master mix. Organize your Master Mix: The first thing is to make sure you didn’t forget anything. The shared information can be so useful for others.īasic Tips When Your PCR Results in No Bands: Provide your own tips in the comment section. For veteran researchers, we’d like to open this up for discussion. And in this article, I’ll provide some tips to help when you encounter a PCR yielding no results. What to do when you get nonspecific binding. So we’re breaking down PCR troubleshooting tips into a series of articles. We understand that for researchers, especially those who are just starting out, PCR can be a nightmare. PCR (Polymerase Chain Reaction), which is so common to the lab, can be very frustrating. ![]()
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