Primer Design Validation

Question

[mikeborg85]

When selecting a commercial primer for a particular organism, is it necessary to validate that the primers can be amplified amongst the genomic DNA of the host organism? Wouldn’t commercial primers typically be validated in this way already? Also, even if I were to design my primers in silico, would I need to validate amplification amongst genomic DNA?

Answer 1

[mark.ziemann]

Yes it is good practice to validate primers on one or a few samples before undertaking your experiment. Things to check are whether the PCR product is the expected size. While the primers are likely tested by the supplier, you want to be certain that it is compatible with the elution buffer all of the other PCR reagents. If you are designing your own primers, you need to check that only 1 product is generated. What is your application? Is it RT-qPCR or something else?