PCR PCR Reagents & Consumables Management 2 — Questions and Answers
Question 1: When performing RT-PCR, why must random hexamer primers be used in the reverse transcription step instead of oligo-dT primers for fragmented or degraded RNA samples?
- Random hexamers prime throughout the RNA, capturing sequences even when poly-A tails are degraded (Correct answer)
- Oligo-dT primers are more expensive and less available
- Random hexamers have higher Tm values than oligo-dT primers
- Oligo-dT primers inhibit reverse transcriptase activity
Correct answer: Random hexamers prime throughout the RNA, capturing sequences even when poly-A tails are degraded
Degraded RNA often lacks intact poly-A tails; random hexamers bind internally throughout the RNA transcript, allowing reverse transcription of fragmented samples.
Question 2: What is the correct storage temperature for most fluorescently labeled TaqMan probes used in real-time PCR to prevent photobleaching and hydrolysis?
- -20°C in the dark (Correct answer)
- 4°C in ambient light
- 37°C to maintain solubility
- Room temperature in amber vials only
Correct answer: -20°C in the dark
TaqMan probes should be stored at -20°C protected from light, as fluorescent dye labels are susceptible to both photo-oxidation and chemical hydrolysis at higher temperatures.
Question 3: Which decontamination reagent should be used to eliminate amplicon carryover contamination from PCR laboratory work surfaces before reaction setup?
- 10% bleach (sodium hypochlorite) solution (Correct answer)
- 70% ethanol only
- DEPC-treated water
- Phosphate-buffered saline (PBS)
Correct answer: 10% bleach (sodium hypochlorite) solution
10% bleach effectively degrades DNA and destroys PCR amplicons on surfaces, preventing carryover contamination; ethanol alone is insufficient for DNA degradation.
Question 4: What is the purpose of including uracil-DNA glycosylase (UDG) and dUTP in a PCR reagent system designed to prevent amplicon contamination?
- UDG degrades any dU-containing carryover amplicons before each new PCR run (Correct answer)
- UDG improves polymerase fidelity during synthesis
- dUTP increases the melting temperature of the amplicon
- UDG increases primer annealing efficiency
Correct answer: UDG degrades any dU-containing carryover amplicons before each new PCR run
The UDG/dUTP system incorporates uracil into amplicons; UDG added before each new run degrades prior amplicons containing dU, preventing false positives from carryover.
Question 5: Which specification should be verified on a Certificate of Analysis (CoA) when receiving a new lot of dNTPs for PCR to ensure reagent quality?
- Purity (≥99%) and absence of nuclease contamination (Correct answer)
- Color of the solution
- Viscosity measurements
- Osmolality of the solution
Correct answer: Purity (≥99%) and absence of nuclease contamination
High purity dNTPs (≥99% by HPLC) and confirmed absence of DNase/RNase are critical quality parameters on a CoA that directly affect PCR performance and reliability.
Question 6: When using optical adhesive sealing films on 96-well PCR plates for real-time PCR, what critical quality issue must be avoided during application?
- Air bubbles between the film and plate wells, which scatter fluorescent signal (Correct answer)
- Applying the film too slowly
- Using a roller applicator instead of hand pressure
- Sealing the plate before adding the master mix
Correct answer: Air bubbles between the film and plate wells, which scatter fluorescent signal
Air bubbles trapped under the optical sealing film scatter or block the fluorescent signal from the wells, causing inaccurate Ct values or well dropouts in real-time PCR.
PCR PCR Reagents & Consumables Management 2
When performing RT-PCR, why must random hexamer primers be used in the reverse transcription step instead of oligo-dT primers for fragmented or degraded RNA samples?