What is “Hot-start” PCR?
“Hot-start” PCR is a method that generally produces cleaner PCR products. Template DNA and primers are mixed together and held at a temperature above the threshold of non-specific binding of primer to template. All the PCR reaction components are added for the extention reaction except one critical reagent (usually the thermostable polymerase). Just prior to the cycling, the missing component is added to allow the reaction to take place at higher temperature. Due to lack of non-specific hybridization of primers to template, the amplified DNA bands tend to be cleaner; the primers don’t have a chance to anneal non-specifically. This method is difficult to do because the tubes must be kept on a 100 degrees C heat block as your work surface. There are ways to avoid this however. One way is to quickly cool the tubes on ice while adding the component mix. You can then heat the tubes on the pre-warmed thermocycler just before adding the last component. This may not always be successful due to
