Thermal Cycling Parameters & Optimization Flashcards

A protocol uses a chemically modified hot-start Taq. What does its initial denaturation step usually need?

Answer: About 10-15 minutes at 95°C to activate the enzyme

Chemically blocked hot-start polymerases need a long high-temperature incubation to remove the blocking groups.

When is a two-step PCR protocol (combined annealing/extension) appropriate?

Answer: When primer Tm values are close to the extension temperature (about 65-72°C)

If primers anneal well near 68-72°C, annealing and extension can happen in one step, which shortens the run.

How is a starting annealing temperature usually chosen for a new primer pair?

Answer: About 3-5°C below the lower primer Tm

Starting just below the lower Tm lets both primers bind while keeping reasonable specificity.

In thermal cycler specifications, what does 'ramp rate' mean?

Answer: The speed of temperature change between steps, in °C per second

Ramp rate is how fast the block heats or cools between setpoints, and it affects total run time.

What is the main purpose of the heated lid on a thermal cycler?

Answer: To prevent evaporation and condensation on the tube caps

Keeping the lid at about 105°C stops water from condensing on the caps, so reaction volume and concentrations stay the same.

Why should denaturation steps not be made much longer than needed?

Answer: Long high-temperature exposure reduces polymerase activity and can depurinate the template

Extended time at 94-98°C shortens enzyme half-life and damages DNA through depurination.

A lab switches from Taq to native Pfu polymerase for a 2 kb amplicon. Which cycling change is usually needed?

Answer: Increase the extension time, since Pfu synthesizes more slowly than Taq

Native Pfu is a slower proofreading enzyme (about 1-2 min/kb), so extension times must be longer.