NeoProof DNA polymerase
Description
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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