2 × Phanta Flash Master Mix is based on the advanced and superior enzyme Phanta Flash Super-Fidelity DNA Polymerase. Through directed optimization of Phanta DNA Polymerase, Phanta Flash Super-Fidelity DNA Polymerase has the features of rapid amplification (4 - 5 sec/kb) while sustaining high yield and fidelity. Compatible with an optimized buffer system, this kit can attain high amplification specificity.
It also has better compatibility with crude samples, templates with uracil and a GC-rich system (primer/template). The kit consists of two types of monoclonal antibodies inhibiting the 5'→3' polymerase activity and 3'→5' exonuclease activity at room temperature, thus enabling it to perform hot-start PCR with better specificity.
It contains all of the required reaction components (Phanta Flash Super-Fidelity DNA Polymerase, optimized buffer and dNTP), excluding primers and templates, thus streamlining the operation process and enhancing the detection efficiency and consistency.
Amplification will generate blunt-ended products, which have compatibility with TOPO cloning kits (Vazyme #C601) and ClonExpress kits (Vazyme #C112/C113/C115).
This product is appropriate for PCR with various templates such as cDNA, genomic DNA, dU-containing DNA, plasmid DNA, crude samples, etc.
- High-quality templates have to be used for successful amplification and products yield.
- The preferred extension time for fragments 10 kb is 4–5 seconds/kb. The extension time is 10 seconds/kb for fragments >10 kb.
- Phanta Flash Super-Fidelity DNA Polymerase has strong proofreading activity. The user has to perform purification prior to dA-tailing for the cloning process.
- Primer Design
- G or C is preferred as the last base at the 3' end of the primer
- Differences in the Tm value of the forward and reverse primers should not be greater than 1 and the Tm value has to be between 55 and 65 (Primer Premier 5 is ideal to determine the Tm value).
- Consecutive mismatches are to be avoided in the last 8 bases at the 3' end of the primer. Hairpin structures at the 3' end of the primer are not preferred.