PCR Reagents & Consumables Management Flashcards

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

Answer: To eliminate DNase and RNase that would degrade template and primers

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.

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?

Answer: Magnesium chloride (MgCl₂) concentration

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.

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?

Answer: No more than 5–10 freeze-thaw cycles

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

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?

Answer: PCR tubes and 96-well plates

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.

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

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

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

Which reagent additive can be included in a PCR master mix to reduce secondary structure in GC-rich templates and improve amplification efficiency?

Answer: DMSO (dimethyl sulfoxide) at 5–10%

DMSO lowers the melting temperature of GC-rich DNA regions by interfering with hydrogen bonding, reducing secondary structures and improving polymerase access to the template.