Producing high yields of good-quality PCR products requires a complex combination of optimal chemistry, primer design, template quality, and cycling parameters. The details of these reaction conditions change for every PCR product, and suboptimal reagent concentrations and annealing times lead to sequence errors, incorrect product sizes, nonspecific products, or a lack of product. Therefore, researchers must optimize their PCR reagents and conditions for every new template and primer set.
The unique features of every DNA template change the PCR chemistry. Researchers should modify their starting template concentration based on the DNA composition and type. For example, a PCR using a genomic DNA template requires a higher template concentration compared to one with a plasmid DNA template. Additionally, the DNA concentration may need to be altered to accommodate different DNA polymerases.
Once a researcher determines the ideal template concentration, they should optimize their reaction’s amplification efficiency by testing the other reaction ...
