Data Analysis & Troubleshooting Flashcards

A conventional PCR gives no band for your samples, but the positive control amplifies strongly. What is the most likely cause?

Answer: The sample template has inhibitors or is degraded

A working positive control rules out reagent and cycler failure, so the problem is likely the sample template.

Your PCR products run as a smear on an agarose gel. Which change is most likely to help?

Answer: Lower the amount of template or the number of cycles

Smears often come from too much template or too many cycles, which cause nonspecific products and over-amplification.

PCR produces the expected band plus several extra nonspecific bands. Which adjustment is most appropriate?

Answer: Raise the annealing temperature or use touchdown PCR

Higher stringency during annealing reduces primer binding to partially matched sites.

A band of the expected size shows up in the no-template control. Which strategy best prevents carryover contamination from earlier PCR products?

Answer: Use dUTP in reactions with uracil-N-glycosylase (UNG) treatment

UNG breaks down uracil-containing amplicons from earlier runs before cycling starts, preventing carryover contamination.

Diluting a sample 1:10 causes it to amplify earlier (lower Ct) than the undiluted sample. What does this suggest?

Answer: The undiluted sample contains PCR inhibitors

Dilution lowers inhibitor concentration, so amplification improves even though there is less template.

A faint band below 100 bp appears in every lane, including the NTC. What is it most likely?

Answer: Primer dimer

Primer dimers are short artifacts that form even without template and run below about 100 bp.

Which reaction component, when too high, most commonly lowers specificity and increases nonspecific products?

Answer: Magnesium chloride

Too much Mg2+ stabilizes mismatched primer-template binding and lowers polymerase fidelity, increasing nonspecific products.