How to avoid contamination during PCR?
Plate loading technology is important. When loading reactions into reaction plates, it is important to do so in a way that reduces cross-contamination. Here are some best practices:Do not let pipette tips pass through other wells or different sample/assay types.
Change pipette tips between replicate groups and sample types.
Create a plate loading guide to keep track of the samples in each well. At the end of a row, check for empty holes in the board.
Add reagents according to your experimental goals. If you are testing multiple samples against the same assay/master mix, pipette the master mix into all wells before adding your samples. This will make dispensing easier and reduce assay contamination since all wells in a given location are testing the same assay. If adding samples first, pipette tips should be replaced between each addition of the master mix to avoid cross-contamination.
If you have many different assays that you will be testing on a small number of samples, it will be easier to dispense your samples first and then add your master mix. Each time a master mix for a different assay is added to the wells, the pipette tips must be changed between each different sample set. Even if the detection method is the same, it is necessary to avoid touching the edge of the well with the pipette tip. If unavoidable, tips must be replaced between each well.
Here are two examples of how to load the plate depending on the experimental setup:
To analyze the expression of two genes in 20 different samples, first dispense the assay mixture into the reaction plate and then add the samples.
To analyze the expression of 20 genes in only two different samples, it would be easier to first dispense the samples and then add the assay master mix.
Regardless of how the plates are loaded, it is essential to avoid cross-contamination.
When loading the plate with a master mix containing the same assay, a multichannel pipette can be used. However, care must be taken to ensure that each hole is loaded with the same amount, and preferably not to touch the edges of the board holes.
Using a splash-proof base helps keep the plate stable during loading and avoids the plate coming into contact with the lab bench surface.
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