4 Effective Ways to Minimize PCR Contamination
Scientists have used polymerase chain reaction (PCR) for over 35 years now, and the process has been refined throughout the years.
However, you can still encounter a few contamination problems. The major causes of PCR contamination are cloned DNA, previous DNA extraction, and carryover contamination. Carryover contamination is when DNA of the same target sequence is generated from a previous experiment, and it is the most common cause of contamination.
Fortunately, you can avoid these problems by applying a few tactics. The following are four ways to minimize PCR contamination.
1. Organize the Laboratory
If your lab is not well organized, samples can easily get mixed and give inaccurate results. Hence, you need to rearrange your lab and have two rooms: pre-PCR and post-PCR locations.
At the pre-PCR area, you will prepare your sample using a manual or an automatic extraction method. Therefore, this room will have preparation lab equipment such as pipettes and pipette tips, centrifuges, tubes, and liquid handling equipment. You will also have the necessary reagents such as chloroform and phenol.
The room should also have a DNA purification kit to remove materials that can affect your experiment. You should also have a refrigerator or freezer to store your samples. You can perform sample preparations in two separate benches to further minimize the risk of contamination.
In the post-PCR room, you will perform all the procedures after the preparation process. Hence, you will have your thermal cycler or PCR machine and electrophoresis apparatus in this room. You also need pipettes tips, tubes, and a mini-centrifuge. Having two designated areas will significantly reduce the chances of contamination.
2. Observe a Unidirectional Workflow
In molecular biology, a unidirectional workflow is when the items and samples of one room can’t be introduced to another room. Therefore, no apparatus should leave the post-PCR room to be used in the pre-PCR one. This also applies to lab coats, eyewear, and gloves.
For instance, test tubes in the post-PCR lab contain amplified DNA segments, referred to as amplicons. If these tubes are used in the pre-PCR room, the amplicons may act as templates in your next experiment, increasing the chances of carryover contamination.
If you have adequate employees, the lab technicians working in the pre-PCR room should not work in the post-PCR area. However, if you have a few employees who must handle all the tasks, they should always change their PPE and disinfect when moving from one room to another.
3. Use PCR-Grade Water
Water is required in PCR to provide a liquid environment for the reaction and a final sample volume. However, the water can introduce contaminants to your sample if it has nucleases.
Therefore, you must use PCR-grade water, which has undergone purification, sterilization, filtration, and autoclaving. All these processes work to remove any contaminants that can affect the integrity of your results.
4. Aliquot the Reagents
Your reagents can also cause PCR contamination. When you collect samples from your reagent container, you may contaminate them if you use an unsterilized tool or a wrong method. This means that you will destroy your reagent, and you will have to buy a new one, which can be costly.
Fortunately, when you divide your reagents, you can store them in smaller tubes and work with one aliquot at a time. This way, you won’t jeopardize your whole reagent. However, you still have to observe proper pipetting methods and use clean tools when collecting the sample.
PCR contamination can lead to false experiment results, so you need to apply these tips. At Revolutionary Science, we offer PCR machines with a touch screen display to help keep tabs on your experiments. If you need a PCR machine or other lab equipment and instruments, contact us today.