AffiTaq® Hot Start - With Buffer - With MgCl2 - With dNTP
Product Overview:
Description: AffiTaq® Hot Start is an advanced DNA Polymerase formulated with a proprietary buffer solution , MgCl2 and dNTPs for superior PCR performance. This specialized variant features a hot-start technology, minimizing non-specific amplification and primer-dimer formation, ensuring enhanced specificity in PCR reactions.
Product Components:
Components | Concentration | AFG-BOR-202 |
---|---|---|
AffiTaq® Hot Start DNA Polymerase | 5 U/μL | 5X (1000U - 200μL) |
AffiTaq® Buffer - dNTP free & Mg2+ free | 10X | 5X (200μL) |
AffiTaq® MgCl2 | 25mM | 5X (200μL) |
AffiTaq® dNTPs | 10mM | 5x (800μL) |
Product Specifications:
Composition:
- DNA Polymerase: AffiTaq® sourced from highly purified recombinant enzymes, offering robust and reliable performance, with an added hot-start feature for minimized non-specific amplification.
- Buffer: A unique proprietary buffer blend optimized for enzyme activity and stability during amplification reactions.
- MgCl2 Concentration: Included at an optimized concentration for enhanced polymerase activity.
- dNTPs: serve as the building blocks for DNA synthesis during PCR reactions, providing the necessary nucleotides for polymerase extension.
Storage Conditions:
- Temperature: Store at -20°C for long-term stability and preservation of enzymatic activity.
- Stability: Maintains stable activity for up to 24 months under recommended conditions.
Performance Characteristics:
Key Features:
- Hot-Start Technology: AffiTaq® Hot Start minimizes non-specific amplification and primer-dimer formation, ensuring enhanced specificity in PCR reactions.
- Robustness and Stability: The hot-start variant maintains high robustness and stability in diverse PCR applications.
- Versatile Usage: Suitable for various PCR applications, including routine PCR, genotyping, and high-throughput PCR.
Applications:
- PCR Applications: Ideal for routine PCR, genotyping, and other molecular biology applications requiring high-quality amplification with minimized non-specific products.
- Sensitivity and Specificity: Demonstrates exceptional sensitivity and specificity across various templates and reaction conditions, improving assay reliability.
Quality Control:
QC Tests Performed:
- Stringent quality control tests include purity assays, activity assays, and specificity assays, guaranteeing consistent performance and reliability.
Quality Assurance:
- Manufactured under ISO-certified conditions, maintaining rigorous quality assurance protocols for batch-to-batch consistency and reliability.
Usage Guidelines:
Recommended Protocol:
- Detailed protocols and guidelines provided for using AffiTaq® Hot Start DNA Polymerase, emphasizing the importance of the initial denaturation step and optimal cycling parameters for superior results.
- Recommended Concentrations: General recommendations for primer and template concentrations provided for various PCR applications.
Handling Precautions:
- Adhere to standard laboratory safety practices when handling enzymes. Avoid repeated freeze-thaw cycles for optimal enzyme activity.
Regulatory Compliance:
Certifications and Compliance:
- Complies with relevant regulatory standards and manufactured in accordance with ISO 9001:2015 guidelines.
- Certified free of contaminants such as endonucleases, exonucleases, and RNases.
Packaging Information:
Packaging Details:
- Available in various packaging sizes to suit diverse laboratory requirements, featuring stringent sealing to prevent contamination.
- Packaging materials ensure stability and integrity during transportation and storage.
Labeling Information:
- Each product is labeled with batch numbers, expiration dates, storage conditions, and specific composition details for traceability and quality control purposes.
Suggested Protocol for PCR Using AffiTaq® Hot Start - With Buffer - With MgCl2 - Without dNTP
Materials Provided:
- AffiTaq® Hot Start DNA Polymerase (5 U/μL)
- AffiTaq® Buffer (10X)
- AffiTaq® MgCl2 (25mM)
- AffiTaq® dNTPs
Materials Required but not provided:
- Primer pairs specific to the target DNA sequence (25)
- Template DNA
- PCR tubes/strips and PCR machine
- Sterile, nuclease-free water
- PCR-grade pipettes and tips
Protocol:
Preparation:
-
Thaw the AffiTaq® Hot Start DNA Polymerase set components on ice.
-
Prepare a master mix for each PCR reaction as follows:
- AffiTaq® Hot Start DNA Polymerase (5 U/μl)
- AffiTaq® Buffer (10X)
- AffiTaq® dNTPs
- MgCl2 (if not included in the buffer, add as specified in the product instructions)
- Sterile, nuclease-free water
PCR Reaction Setup:
- Set up the PCR reaction tubes/strips on ice.
- Add the following components to each PCR tube:
Components | Concentration | Volume per Reaction | Final Concentration |
---|---|---|---|
AffiTaq® Buffer - dNTP free & Mg2+ free | 10X | 5 μL | 1X |
AffiTaq® dNTPs | 10 mM | 1 μL | 200 μM |
AffiTaq® MgCl2 | 25mM | 2 to 8 μL | 1 to 4 mM |
AffiTaq® Hot Start DNA Polymerase | 5U/μL | 0.25 to 0.5 μL | 1.25 to 2.5 U |
Primer Mix | 25 | 2 μL | 1X |
Template | - | - | Less than 1μg/Reaction |
ddH2O | - | Adjust to 50 μL | - |
- Mix the contents gently by pipetting and briefly spin down the tubes to collect all the liquid at the bottom.
PCR Cycling Conditions:
-
Start the PCR machine and set the following cycling parameters:
- Initial Denaturation: 95°C for 2-5 minutes (1 cycle)
- Denaturation: 95°C for 15-30 seconds
- Annealing: [Optimal temperature]°C for 15-30 seconds
- Extension: 72°C for [Extension time based on product guidelines] seconds
- Final Extension: 72°C for 5-10 minutes (1 cycle)
- Hold at 4-10°C
-
Run the PCR according to the specified cycling parameters for the recommended number of cycles for your specific target.
Post-PCR Steps:
- Analyze the PCR products using agarose gel electrophoresis or other appropriate methods.
- Store the remaining PCR products at -20°C for further analysis or downstream applications.
Notes:
- Adhere to aseptic techniques and wear gloves to prevent contamination.
- Adjust annealing temperature and extension time, if necessary, for specific primer-template combinations.
- Always refer to the provided AffiTaq® Hot Start DNA Polymerase set instructions for optimal conditions based on your experiment.