Thermal Cycling Parameters & Optimization Flashcards

A PCR run produces several nonspecific bands in addition to the target. Which thermal cycling adjustment should be tried first?

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

A higher annealing temperature increases stringency, so primers bind less often to partially matched sites.

In touchdown PCR, how is the annealing temperature programmed?

Answer: Started above the primer Tm and lowered by about 0.5-1°C per cycle

Early high-stringency cycles favor the specific product, which then outcompetes nonspecific products in later cycles.

What is the main advantage of a gradient thermal cycler during assay optimization?

Answer: It tests a range of annealing temperatures across the block in one run

A gradient block holds different columns at different temperatures, so the best annealing temperature can be found in one experiment.

A template has 72% GC content and gives weak or no amplification. Which change is most likely to help?

Answer: Raise the denaturation temperature or time and add DMSO or betaine

GC-rich DNA has stronger strand pairing and secondary structure, so it needs harsher denaturation and helper additives.

Using standard Taq polymerase, about how long should the extension step be for a 3 kb amplicon?

Answer: About 3 minutes

The usual rule for Taq is about 1 minute of extension per kilobase of product.

Besides finishing incomplete products, what is a key benefit of a 5-10 minute final extension at 72°C when using Taq?

Answer: It adds 3' A-overhangs needed for TA cloning

Taq's terminal transferase activity adds a single 3' adenine, and a long final extension lets more products get this overhang.

What is a common result of running too many cycles (e.g., 40-45) in endpoint PCR?

Answer: Smearing and buildup of nonspecific products after the plateau

Once reagents run low, extra cycles mostly amplify mispriming products and artifacts, which shows up as smears.