Free PCR Data Analysis & Troubleshooting Questions and Answers

Question 1

What does a clear single band on a gel indicate after PCR?

Accepted Answer

Successful target amplification

Answer

A clear, single band on an agarose gel at the expected size after PCR indicates that the reaction successfully amplified the specific target DNA sequence. This result confirms that the primers were specific, the reaction conditions were optimal, and the desired product was generated efficiently. It also suggests the absence of significant non-specific amplification or primer dimers.

Question 2

What might cause no bands to appear in a PCR gel?

Accepted Answer

Missing enzyme or wrong temperature

Answer

The absence of any bands on a PCR gel strongly suggests that the amplification reaction failed. This can be caused by critical missing components, such as the DNA polymerase enzyme which is essential for DNA synthesis, or by incorrect temperature settings. Improper temperatures can prevent denaturation, annealing, or extension from occurring effectively, thus halting the amplification process.

Question 3

What does a smear on the gel indicate?

Accepted Answer

Degraded DNA or over-cycling

Answer

A smear on an agarose gel after PCR typically indicates a range of DNA fragment sizes rather than a distinct, specific band. This can result from degraded template DNA, which leads to fragmented amplification products. Alternatively, over-cycling can cause the accumulation of non-specific products, primer dimers, and even degradation of the desired product, all contributing to a smeared appearance.

Question 4

Which factor helps confirm primer specificity?

Accepted Answer

Melt curve analysis

Answer

Melt curve analysis, commonly used in real-time PCR, involves slowly increasing the temperature and monitoring the dissociation of double-stranded DNA. Each specific PCR product has a unique melting temperature (Tm) at which its strands separate. The presence of a single, sharp peak at the expected Tm confirms the specificity of the primers and the absence of non-specific products or primer dimers.

Question 5

Why is a negative control used in PCR?

Accepted Answer

To check for contamination

Answer

A negative control in PCR contains all reaction components except the template DNA, with water replacing the template. If amplification occurs in this control, it indicates contamination of reagents, primers, or equipment with exogenous DNA. This contamination could lead to false positive results in experimental samples, making the negative control essential for validating results.

PCR - Polymerase Chain Reaction Certification Practice Test

PCR - Polymerase Chain Reaction Certification Practice Test

Free PCR Data Analysis & Troubleshooting Questions and Answers