4 PCR Tips When Encountering Nonspecific Binding:
by Katharine Martin
One day your PCR works; then when you repeat it, you get no results, and when you try yet again, you get nonspecific binding. It’s these situations that drive you to superstitious rituals and prayers to the PCR gods for mercy. Unfortunately, divine forms of troubleshooting yield few results.
Four important tips when you encounter nonspecific binding include aliquoting, using negative controls, increasing your annealing temperatures and doing touch-down PCR. But each tip needs further explanation.
For a veteran in life science research, PCR has become second nature; however, I have seen undergraduate and graduate students highly stressed out about PCR. I have heard them utter to one another, “I’m about to see if this works. Wish me luck.” I knew one undergraduate student who struggled for an entire semester trying to make PCR work only to end up switching majors. After investing years into scientific coursework and research, we don’t want it to come to that. Instead, we’re here to help.
Rather than having a huge troubleshooting article with every single PCR tip known to scientists, I’ll break this into a series: nonspecific binding, no results, smearing, weak results and contamination.
1. Aliquot Aliquot Aliquot:
If you remember the article about fridge & freezer organization, certain areas of your upright freezer have a greater risk of unintentionally exposing your reagents to freeze-thaws. Outside of the freezer, you also run the risk of contamination to your reagents that might degrade them. Simply put, protect your supplies. Aliquot DNTPs, primers, etc. Only move your working vials into easily accessible freezer boxes. Store the rest of your stock in a more protected area at -80 °C.
2. Negative Controls:
This is a must in every PCR setup. In fact, my mentor made it a practice to set up the first two tubes and last two tubes to be positive and negative controls. This let both he and I see if contamination was ever a reason for a bad PCR. And it let us see whether or not it occurred throughout the whole process.
3. Increase Annealing Temp:
By increasing the annealing temperature, you’re driving specificity. In general, you want to use an annealing temperature that is 5 °C lower than the Tm of your primers.
4. Touch-down PCR:
In this process, the first stages of PCR should have a high annealing temperature, even higher than the estimated Tm of your primers. Following cycles have incrementally lower annealing temperatures. This gradual adjustment stops when you have reached the calculated annealing temperature of your PCR primers. The tricky part will be deciding on the incremental decreases you want to use. With the higher annealing temperature being used at the beginning, the resulting sequence will be the most specific and able to out-compete nonspecific results.
Stay tuned for more PCR tips in the following article. We hope that some of these tips begin to help you attain the results you’re hoping for.
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