Delivers 50× higher fidelity than Taq DNA polymerase with blunt-end amplification. Suitable for routine PCR, colony PCR, challenging templates, and high-GC content PCR.
Cat. No. :
Y101312-1MLBrand :
YanbiotechProduct Origin :
ChinaVolume :
1 mLProduct Information：
2x Fast High Fidelity PCR Master Mix is a ready-to-use PCR premix containing modified ultra-high fidelity DNA polymerase，dNTPs and optimized PCR reaction buffer. PCR amplification can be carried out by adding only template, primer and ddH2O so that the concentration of Mix solution is 1×. Strong amplification ability, fast amplification speed, extension speed up to 5-15 s/kb, with ultra-high fidelity, the fidelity is 50 times that of Taq DNA Polymerase and 6 times that of Pfu DNA Polymerase, and the amplification product is flat-ended. Loading Dye has been added to the product, and the amplification product can be directly used for agarose gel electrophoresis detection. This product is suitable for PCR amplification of conventional PCR, colony PCR, complex templates and templates with high GC content.
Instructions for Use:
Recommended PCR reaction system:
| Component | 20 μL | 50 μL | Final Concentration |
| Templatea | Variable | Variable | as required |
| Forward Primer (10μM)b | 0.8 μL | 2 μL | 0.4μM |
| Reverse Primer (10μM)b | 0.8 μL | 2 μL | 0.4 μM |
| 2×Fast High Fidelity PCR Master Mix | 10 μL | 25 μL | 1× |
| (DMSO, optional)c | (0.6 μL) | (1.5 μL) | (3%) |
| ddH2O | Add to 20 μL | Add to 50 μL |
a: If the template is plasmid or from bacteriophage, 50ng-5pg added in 50 μL rnx is recommended. If tempalted is genomic DNA, 250ng-50ng added in 50 μL rnx is recommended. If template is cDNA, dilute your original cDNA by 2-100 times, and the volume of diluted cDNA added to reaction is no more than 10% of total reaction volume.Higher amounts of template tend to lead to non-specific amplification. Lower amounts of template tend to lead to low PCR amplification efficiency.
b: The range of final primer concentration used is 0.2-1.0 μM，and 0.4 μM is recommended. Too little primer may result in low or no amplification yield, and too much primer may result in non-specific amplification.
c: For high GC content template, DMSO with no more than 10% total volume can be added.
Recommended PCR amplification system conditions:
| Step | Temperature | Volume | Cycles |
| Initial Denaturationa | 98℃ | 30 s | 1 |
| Denaturation | 98℃ | 15-30 s | 25-35 |
| Annealingb | 50-72℃ | 15-30 s | |
| Extensionc | 72℃ | 5-15 s/kb | |
| Final extension | 72℃ | 5-10 min | 1 |
| Hold | 4-16℃ | Forever |
a: A pre-denaturation time of 30 s is suitable for most conventional templates; complex templates can be denatured for up to 2 min.
b: For amplification of complex templates, containing concatenated primers, the annealing time can be extended up to 30 s.
c: The recommended extension speed of plasmid and other simple templates is 5-10 s/KB; The recommended extension speed of conventional genomic template is 10-15 s/KB; Complex template 15-30 s/KB.
Notice:
1. Thoroughly thaw and mix before use. It can be kept at 4℃ for at least 2 weeks to avoid freeze-thaw cycles.
2. For your safty and health, please wear safety glasses, gloves, or protective clothing.
3. For Research Use Only.
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