Imagine that you accurately set up the PCR test, anxiously waited for it as it ran, and then stood back for the big reveal. Now imagine that instead of seeing what you wanted, you see a big mess. Rather than a nice clean gel, you notice extra bands and even bands in your negative control. This is what we call a classic case of PCR contamination.
Although there are a lot of reasons why this could happen, you should first look into your equipment and see if they are free of any kind of contaminants. Even so much so as a dust particle in the Rainin LTS Filter Tips could lead to a failed test.
Now, how do you fix the PCR contamination and avoid it from happening in the future? Well, we are here to tell you just that. Keep reading to find out!
What Is PCR Contamination?
Aerosolized PCR products are the main source of PCR contamination. This usually happens when you open a pipette or tube and amplified droplets are created. Once that happens, they tend to travel very rapidly because of their small size. In no time, these droplets will be all over your equipment and work-bench and eventually will find a way into your PCR to be further amplified. This is essentially what PCR contamination implies.
But how do you know if you have PCR contamination? The answer is negative controls.
Run Negative Controls
Running negative controls every time you run a test is an extremely critical step. Every lab worker knows it but sometimes, feeling lazy, we skip out on them. We make up excuses such as, “the experiment always works,” “we do not have enough time,” and “we could save on regents.” But people with years of experience in labs suggest that running negative control is highly imperative, as it saves time in the long run and also saves precious resources. (Redig)
A PCR negative control is technically similar to a PCR master mix but rather than mixing in the template, you use water. Once you do that, it should result in no PCR product and an empty gel lane. Although if you do get a band, you know you have a contaminated PCR.
Identify Your Contamination Source
What could be the source of this contamination? Basically, anywhere. However, it could be originating from one of two places: 1) your laboratory environment (pipettes, tips, hands, benchtop, centrifuge, etc.) or 2) your reagents (polymerase, nucleotides, buffer, water, and other reagents).
Rule Out Your Laboratory Environment
When looking for PCR contamination, the first step is to eliminate all probable environmental sources. To accomplish this, you should use a ten percent DNA-away or bleach solution to wipe down your:
- Thermocycler lid and buttons
You should also buy new:
- Unopened sterile PCR tubes
- Unopened filter tip boxes
In addition to that, you should also assemble your PCR reaction in the correct manner. Here are the essential steps you should carry out.
- Wear a lab coat that is dedicated to your work. This isn’t the same coat you’ll be wearing when you’re examining your PCR findings. It should also not be worn near open tubes with a PCR product that has been amplified.
- Replace your gloves on a regular basis. If you need to leave your PCR setup and specialized equipment for any reason, such as getting more reagents, answering the phone, or even using a pen, make sure to replace your old gloves with new ones before returning to the setup.
Rule Out Reagents
It’s time to double-check your reagents now that you’ve eliminated any fresh PCR contamination from your PCR assembly. This entails replacing each of your old reagents with a fresh, unopened reagent and running the negative control tests again in a systematic manner.
Now that you are aware of how PCR contamination spreads and how to avoid them, you are ready to run your PCR test without anything coming in your way.
So, if you are in search of PCR plates and other molecular biology products, get in touch with MBP inc. We promise our customers the best equipment for their experiments at the best price. Give us a call and place your order now!
Redig, Jennifer. “Clean Up Your Act! How To Clean Up PCR Contamination.” Bitesize Bio, 1 January 2015, https://bitesizebio.com/20773/clean-up-your-act-how-to-clean-up-pcr-contamination/. Accessed 2 June 2022.