A PCR test is one of the most commonly performed techniques in life science laboratories. The bad news is that it is easy to make mistakes, such as processing the wrong sample, forgetting to add a reagent, and creep up when processing large batches of samples. The following are some of the things you should do in order to avoid PCR errors.
First of all, you need to setup a sterile environment. Presence of contaminants can lead to false negatives or positives, which can cause you to start second guessing your results. These contaminants can come in many forms, such as foreign DNA to chemical inhibitors which reduce the efficiency of the reaction. In order to minimize contamination of your PCR samples and reagents, you can use sterile filter tips that will prevent any contamination from your pipette from bleeding into your samples.
You also need to dedicate a clean set of equipment, including pipettes and racks, solely for PCR use to ensure minimal carryover of particles from one area of your lab to another. Other things you can do in order to minimize contamination include setting up a reserved space for all your PCR reactions to further reduce particle contamination, applying ethanol, bleach, and RNaseOUT to your bench, pipettes, and racks to rinse off contaminants, and using gloves at every step and change them often.
Another thing you can do in order to avoid PCR test errors is check template purity and concentration. N matter how clean your bench and equipment are, it is still important to verify the purity of your samples before running them. A lot of analyzers can measure both the concentration and purity of your DNA simultaneously. You need to make sure that your 260 nm/280 nm absorbance ratio is at least 1.8.
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