PCR PCR Reagents & Consumables Management

Question 1

What is the primary reason PCR-grade water (nuclease-free water) must be used instead of regular distilled water in PCR reactions?

Accepted Answer

To eliminate DNase and RNase that would degrade template and primers

Answer

PCR-grade water is treated to remove nucleases (DNase/RNase) that would degrade the DNA template or RNA in RT-PCR reactions, preventing false negatives.

Question 2

Which buffer component in a standard PCR master mix is critical for stabilizing Taq DNA polymerase activity and must be verified when troubleshooting failed reactions?

Accepted Answer

Magnesium chloride (MgCl₂) concentration

Answer

MgCl₂ acts as a cofactor for Taq polymerase and affects primer annealing; its concentration must be optimized (typically 1.5–2.5 mM) as too little or too much inhibits amplification.

Question 3

When storing a prepared PCR master mix containing dNTPs and primers at -20°C, what is the recommended maximum number of freeze-thaw cycles before reagent degradation becomes a concern?

Accepted Answer

No more than 5–10 freeze-thaw cycles

Answer

Repeated freeze-thaw cycling degrades dNTPs and primers through hydrolysis and deamination, with performance typically declining noticeably after 5–10 cycles.

Question 4

Which class of PCR consumables must be certified as 'low-retention' or 'thin-walled' to ensure accurate thermal transfer and prevent sample loss during reaction setup?

Accepted Answer

PCR tubes and 96-well plates

Answer

Thin-walled, low-retention PCR tubes and plates ensure efficient heat transfer between the thermocycler block and the reaction mix and minimize sample adhesion to the plastic walls.

Question 5

What is the purpose of including a 'hot-start' enzyme component in a PCR master mix?

Accepted Answer

To prevent non-specific amplification and primer-dimer formation at room temperature during setup

Answer

Hot-start enzymes are inactive until activated by heat, preventing the polymerase from extending non-specifically annealed primers during room-temperature reaction setup.

PCR - Polymerase Chain Reaction Certification Practice Test

PCR - Polymerase Chain Reaction Certification Practice Test

PCR PCR Reagents & Consumables Management