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PCR Contamination Troubleshooting Test for Microbiologists

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This exam was created by a large language model. Please use it with caution, as inaccuracies may be present. Always verify critical information.

Standard PCR Protocol and Problem Scenarios

Standard PCR Protocol

You are performing a routine PCR amplification of a 500 bp gene target from bacterial genomic DNA. The following protocol is standard in your lab:

PCR Reaction Components (per 25 µL reaction):

Nuclease-free Water: Up to 25 µL
5X PCR Buffer: 5 µL
10 mM dNTP Mix: 0.5 µL (final 200 µM each)
10 µM Forward Primer: 0.5 µL (final 200 nM)
10 µM Reverse Primer: 0.5 µL (final 200 nM)
Taq DNA Polymerase (5 U/µL): 0.2 µL (final 1 U)
Template DNA: 1-5 µL (e.g., 50-200 ng genomic DNA)

Negative Control (NTC): A reaction set up identically but replacing Template DNA with an equal volume of Nuclease-free Water. Positive Control (PC): A reaction set up with known good template DNA and primers.

Thermocycling Conditions:

Initial Denaturation: 95°C for 5 minutes
Cycling (30 cycles):
- Denaturation: 95°C for 30 seconds
- Annealing: 55°C for 30 seconds
- Extension: 72°C for 1 minute (for a 500 bp product)
Final Extension: 72°C for 7 minutes
Hold: 4°C

Gel Electrophoresis Conditions:

1.5% Agarose gel, run at 100V for 45 minutes, stained with Ethidium Bromide (EtBr) or a comparable nucleic acid stain. Expected product size: 500 bp.

Scenario 1

After running the PCR and subsequent gel electrophoresis, you observe the following results:

The negative control lane (NTC - no template control) clearly shows a single band of the exact same size (500 bp) as the expected PCR product.
All other lanes (samples and positive control) show the expected 500 bp band with good intensity.
The DNA ladder is clear and shows proper migration.

Based on Scenario 1's observations, what is the most likely contamination source?

Select one option

Contamination of dNTPs

Cross-contamination from previously amplified PCR product

Insufficient denaturation time

Primer dimer formation

Considering the identified source in Scenario 1, what is the most appropriate immediate troubleshooting step?

Select one option

Increase annealing temperature for the next run.

Prepare a new master mix with fresh Taq polymerase.

Decontaminate pipettes and workspace with 10% bleach/DNAse, use aerosol-barrier tips, and prepare setup in a dedicated PCR clean area.

Re-design primers to avoid secondary structures.

Explain your reasoning for selecting the most likely contamination source and the most appropriate troubleshooting step for Scenario 1.

Scenario 2

After PCR and gel electrophoresis, you observe:

Weak or no expected product band (500 bp) in all sample and positive control lanes.
The negative control lane (NTC) also shows a faint, diffuse smear across a wide range of sizes (not a distinct band).
The DNA ladder is sharp and clear.

Based on these observations, what is the most likely contamination source?

Select one option

Degraded template DNA in samples

Contamination of a bulk reagent (e.g., nuclease-free water or PCR buffer) with non-specific DNA or inhibitors

Incorrect primer concentration

Thermocycler malfunction

Considering the identified source in Scenario 2, what is the most appropriate immediate troubleshooting step?

Select one option

Increase the number of PCR cycles.

Prepare a completely new master mix using fresh, unopened aliquots of all bulk reagents (water, buffer, dNTPs).

Run a positive control with known good template.

Increase template DNA concentration.

Explain your reasoning for selecting the most likely contamination source and the most appropriate troubleshooting step for Scenario 2.

Scenario 3

Following PCR and gel electrophoresis, you observe:

All sample lanes and the positive control show the expected 500 bp product.
The negative control lane (NTC) shows a band at the expected 500 bp product size, and also several additional faint, larger bands that do not correspond to the expected product.
These additional, larger bands are sometimes visible faintly in sample lanes as well.

Based on these observations, what is the most likely contamination source?

Select one option

Cross-contamination with genomic DNA from the laboratory environment or user

Taq polymerase degradation

Low dNTP concentration

Overly specific primers

Considering the identified source in Scenario 3, what is the most appropriate immediate troubleshooting step?

Select one option

Reduce extension time.

Perform a hot-start PCR.

Use a UV crosslinker to treat the lab space and reagents (if applicable) and rigorously clean surfaces; ensure all reagents are handled in a dedicated clean area with proper PPE.

Increase annealing temperature.

Explain your reasoning for selecting the most likely contamination source and the most appropriate troubleshooting step for Scenario 3.