Conditions for annealing and extension

Determine the optimum annealing temperature experimentally by varying temperatures in 2

increments over a range of 45 to 68. To carry out the combined anneal-extension at 68

(two step PCR), the recommended time setting is 30 seconds to 1 minute per one kilobase.

When temperature is set below 68, longer time will be required.

An annealing temperature that is too high generates no amplification products, while a temperature

that is too low enhances non-specific reactions. An extension time that is too short generates

no amplification products or dominantly non-specific, short products, while too long extension time

causes diffusely smeared electrophoresis bands.

As both TaKaRa Ex Taq and TaKaRa LA Taq show sufficient activity at 60 to 68.

Shuttle PCR (Two temperature PCR) can be conducted, by the anneal-extension temperature

within this range.

When long PCR amplification is required, a significant increase in amplification efficiency may be

expected by employing an Autosegment Extension Method.  Chapter 5. Q4