Thermal Cycling Parameters & Optimization Flashcards

A PCR gives no product, but the reagents and template are confirmed to be good. Which cycling change is a reasonable first step?

Answer: Lower the annealing temperature in 2-3°C steps

If the annealing temperature is too stringent, primers may not bind well enough, so lowering it gradually helps.

A strong band under 100 bp appears in both samples and the no-template control. Which change best reduces it?

Answer: Use hot-start polymerase and raise the annealing temperature

Primer-dimers form at low temperatures before or during cycling, and hot-start enzymes plus higher stringency reduce them.

What is the main purpose of nested PCR?

Answer: To increase specificity by using a second round with internal primers

Only the correct first-round product contains the internal primer sites, so nonspecific products are rarely amplified again.

With 100% efficiency, about how many copies come from one template molecule after 30 cycles?

Answer: About 1 billion

Copy number doubles each cycle, so 2^30 is about 1.07 × 10^9.

Which factor contributes to the plateau phase of PCR?

Answer: Depletion of primers and dNTPs and loss of enzyme activity

Late in cycling, used-up reagents, aging enzyme, and product strands re-pairing with each other limit further amplification.

Identical reactions in edge wells of a 96-well block keep giving lower yields than center wells. What is the most likely cause?

Answer: Uneven temperature across the thermal block

Poor block uniformity, often at the edges, means some wells don't reach the right temperatures, so the instrument should be checked with a temperature verification kit.

Why is a 4°C final hold usually limited to short-term storage of PCR products?

Answer: Long holds wear the Peltier unit and are not a substitute for proper storage

Running the block at 4°C overnight adds strain and condensation risk, so products should be moved to a fridge or freezer.