PCR Data Analysis & Troubleshooting 2

Question 1

In a qPCR run, Sample A has a Ct of 22 and Sample B has a Ct of 28 for the same target. What does this indicate?

Accepted Answer

Sample A had more starting template than Sample B

Answer

Ct is inversely related to starting template quantity, so a lower Ct means more initial target.

Question 2

A standard curve has a slope of -3.32. What amplification efficiency does this correspond to?

Accepted Answer

About 100%

Answer

Efficiency = 10^(-1/slope) - 1, and a slope of -3.32 gives about 100% (perfect doubling each cycle).

Question 3

A standard curve gives a slope of -3.9. What is the most likely explanation?

Accepted Answer

Efficiency is about 80%, suggesting inhibition or a poor assay design

Answer

A slope of -3.9 corresponds to about 80% efficiency, below the usual 90-110% range, which points to inhibitors or a suboptimal assay.

Question 4

A SYBR Green melt curve shows one peak at 84°C and a second, smaller peak at 74°C. What does the 74°C peak most likely represent?

Accepted Answer

Primer dimers or another short nonspecific product

Answer

Short products such as primer dimers melt at lower temperatures than the specific amplicon.

Question 5

In a SYBR Green assay, the no-template control (NTC) amplifies at Ct 37 and its melt peak is well below the target Tm. What is the best interpretation?

Accepted Answer

Primer-dimer formation in the NTC

Answer

Late NTC amplification with a low-Tm melt peak is typical of primer dimers, not contaminating target.

PCR - Polymerase Chain Reaction Certification Practice Test

PCR - Polymerase Chain Reaction Certification Practice Test

PCR Data Analysis & Troubleshooting 2