PCR Data Analysis & Troubleshooting 3

Question 1

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

Accepted Answer

The sample template has inhibitors or is degraded

Answer

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

Question 2

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

Accepted Answer

Lower the amount of template or the number of cycles

Answer

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

Question 3

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

Accepted Answer

Raise the annealing temperature or use touchdown PCR

Answer

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

Question 4

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

Accepted Answer

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

Answer

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

Question 5

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

Accepted Answer

The undiluted sample contains PCR inhibitors

Answer

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

PCR - Polymerase Chain Reaction Certification Practice Test