Description
Product Features
- Ultra-high accuracy: Accuracy is 128 times that of Taq DNA Polymerase.
- Faster amplification: The limit speed can reach 0.5 seconds/kb, efficiently amplifying most fragments in 30 seconds/kb.
- Longer amplification: Effective amplification length for simple templates like plasmids and λ DNA is up to 40 kb, for genomic DNA is up to 20 kb, and for cDNA is up to 10 kb.
- Broad adaptability: Suitable for the amplification of various GC content fragments, super-tolerant to PCR inhibitors, and can be used for direct PCR of various samples.
- Simple process: Pre-mix the dye to minimize experimental steps.
Key Data
- Excellent high GC amplification capacity
654 bp, 900 bp, 800 bp, 1,200 bp, 1,400 bp, and 426 bp target fragments were amplified using human genome as a template, and the GC content of each amplicon was over 68%.
- Reliable amplification accuracy
- Stable raw amplification capability
Components Components P515-01 P515-02 P515-03 2 × Phanta Max Master Mix 1 ml 5 × 1 ml 15 × 1 ml
Storage Store at -30 ~ -15°C for up to 18 months and transport at ≤0°C. ▲ Avoid repeated freeze-thaw cycles.
FAQs
S1: Low amplification efficiency and no amplified bands in the test group.
A1: (1) Primers Check if synthetic primers are degraded due to inappropriate storage; Review primer design and use BLAST to examine primer specificity or redesign primers if necessary. (2) Template The template should be freshly prepared double-stranded DNA, as long-term storage or repeated freeze-thaw cycles can lead to DNA breakage, nicking, or degradation. If the template has an excessively high GC content, add PCR Enhancer (Cat. No. P021) to effectively reduce the melting temperature. Sample templates may contain inhibitors, so it is recommended to reduce the template concentration (use after dilution; if a plant leaf is used, make sure the plant is not rich in polysaccharides or polyphenols, take a fresh, young leaf and trim it to the size of the yellow pipette tip). If cDNA is used, check the purity and integrity of the RNA used for reverse transcription. (3) Enzyme The enzyme used in the reaction has been inactivated. Repeat the experiment with fresh enzyme or enzyme from another source. (4) Amplification system The reaction system is improperly prepared. Repeat the experiment with the correct system. (5) Reaction program Check if the denaturation temperature is correct and if the displayed temperature in the PCR system matches the actual temperature. If the temperature is too high, the enzyme will quickly become ineffective in the first few cycles. If it is too low, the template will not fully denature. If using yeast as a template, the initial denaturation time can be extended up to 10 minutes. If the annealing temperature is not suitable, determine the appropriate annealing temperature by testing the annealing temperatures’ gradient. If the target fragment is long, try the touchdown PCR program. Check if the extension time is sufficient.
S2: Faint amplified bands.
A2: (1) Primers Check if synthetic primers are degraded. (2) Template Check the quality of the template. Long-term storage or repeated freeze-thaw cycles can lead to DNA breakage, nicking, or degradation. Use freshly prepared double-stranded DNA as the template. If the template is depleted, use serial dilutions of the first amplification product as the template for subsequent amplifications. (3) Reaction program Try the touchdown PCR program; Increase the extension time and the number of cycles.
S3: Weak specificity of amplification or non-specific products.
A3: (1) Primers Optimize primer design if it is not optimal. If primers are binding to non-specific regions or forming dimers, optimize by reducing primer concentration or, if necessary, redesigning primers. (2) Template Prepare a new template if the template is impure or contaminated. If the template is degraded or too much template is used, examine the integrity and concentration of the template by electrophoresis and, if necessary, purify the template again. Refer to the User Manual for template input amounts. (3) Reaction program The reaction program is not optimal. If there are various non-specific bands smaller than the target band, increase the annealing temperature and decrease the number of cycles. If there are various non-specific bands larger than the target band, reduce the extension time and the number of cycles.
S4: Amplification products smear or overlap when run on a gel.
A4: (1) Gel Ensure the gel is fully melted during preparation. (2) Primers Check if the primers are degraded. (3) Template The quality of the template is low or excessive. Examine the integrity and concentration of the template by electrophoresis and prepare a new template if necessary. Refer to the User Manual for template input amounts. If the target band is long and cDNA is used as the template, check the purity and integrity of the RNA used for reverse transcription and repeat the reverse transcription process without random primers. (4) Reaction program The reaction program is not optimal. If the annealing temperature is not suitable, determine the appropriate annealing temperature by testing the annealing temperatures’ gradient. Try the touchdown PCR program.
S5: Amplification products in the No Template Control (NTC) group.
A5: (1) Problematic primer design The amplified sequence is homologous to a non-target sequence. Therefore, non-target sequences are also amplified in PCR. (2) If the band corresponding to the amplification product is the same size as the target bands, this indicates contamination. Repeat the experiment with a fresh mixture, water, or primers. Prepare the reaction system in an ultra-clean workbench to minimize aerosol contamination. (3) To prevent cross-contamination by large fragments of the genome or target sequence, carefully follow steps to prevent the loading pipette from pipetting or spilling the target sequence when using the loading pipette. To reduce or eliminate contamination due to small nucleic acid fragments in the air, use the nested PCR method.
S6: Poor quality of high-quality polymerase leading to mutations.
A6: Mutations in high-quality PCR can be analyzed in the following ways:
(1) Mutations in the template. Check if the template has undergone mutation. If it is a plasmid, send it for sequencing. If it is cDNA, repeat the test; if mutations persist, there may be issues with the cDNA. In this case, it is recommended to prepare new cDNAs after checking the integrity and purity of the RNA.
(2) Mutations may result from prolonged exposure of the template to UV light. Avoid prolonged exposure to UV light during gel extraction.
(3) The gene sequence is inconsistent with the NCBI sequence. Resubmit the DNA for sequencing. If the result remains the same, it means that the sequencing may be inconsistent with those listed on NCBI.
(4) Similar recombinant sequences lead to incorrect recombination.
S7: When using cDNA as the template for high-quality PCR, how many templates should be entered?
Answer7: The recommended cDNA template input amount is 1–5 μl (should not exceed 1/10 of the total volume of the PCR system). Determine the appropriate input amount by testing different template amounts within this range.
S8: Do amplification products obtained with high-quality polymerases have poly(A) tails? How are poly(A) tails added?
Answer8: (1) Amplification products obtained with high-quality polymerases do not have poly(A) tails. Try Vazyme’s Seamless Cloning Product C601 for subsequent cloning; Add a poly(A) tail using a universal Taq polymerase for TA cloning:
Poly(A) Tailing Method: 10 ul system containing only Taq polymerase:
1 ul 10× Taq Buffer (containing MgCl2)
0.5 ul 10 mM dNTP
0.2 ul Taq DNA polymerase (without primers)
1 – 7 µl purified PCR fragments
Add ddH2O to 10 ul
Taq Mixture System:
2× Taq Master Mix 5 μl
Purified PCR fragments
ddH2O ]
Reaction conditions: 72°C 10 min.
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