NeoProof DNA polymerase

Product Components and Storage

• Supplied with a 10× Reaction Buffer and 5× Stabilizer Solution
• Recommended storage at -20 °C in a constant temperature freezer
• Stability until the expiry date if stored as specified
• Unit definition: incorporation of 10 nmoles of dNTPs in 30 minutes at 72 °C

PCR Protocol

• Standard protocol provided as a general guideline for PCR amplification
• Guidelines for reaction setup, including component order and assembly on ice
• Cycling parameters for denaturation, annealing, and extension
• Gel electrophoresis for analyzing PCR products

PCR Design Considerations

• Designing PCR primers with appropriate length (15-30 bases)
• Recommendations for primer sequences to improve PCR yield and avoid degradation
• Guidelines for primer GC content and avoidance of internal secondary structure
• Recommendations to prevent primer-dimer formation and non-specific primer annealing
• Importance of similar melting temperatures (Tm) for both primers

DNA Template

• Recommended amounts of starting material based on quality and complexity
• Guidelines for using genomic DNA templates and cDNA synthesis reaction templates
• Caution against exceeding 10% of the final PCR reaction volume for cDNA templates

Enzyme Concentration

• Recommended enzyme concentration of 1.25 U (0.5 μL) in a 50 μL reaction
• Higher enzyme volumes (up to 2.5 U) for amplifying abundant templates (>50 ng gDNA)
• Dilution options for convenience during PCR assembly

Quality Control Assays

• Purity evaluation through SDS-PAGE and Coomassie Blue staining
• Evaluation of genomic DNA contamination through PCR
• Nuclease assays to check for nicking or cutting of nucleic acids
• Functional assay using different-sized DNA fragments from human genomic DNA