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FlashPfu DNA Polymerase 

TonkBio FlashPfu is a DNA polymerase for high-fidelity PCR. By fusing Pfu with a high affinity double-stranded DNA binding domain, we have dramatically increased its processivity and extension rate. FlashPfu DNA polymerase is 50-fold more accurate than Taq polymerase and 6-fold more accurate than Pfu polymerase.
Cat. No. Size Price Quantity  
TB10011A 100 U USD 99
TB10011B 250 U USD 169
TB10011C 2×250 U USD 269
Tel: +1 (973) 789-8318Email: [email protected]

Reagents Supplied

  • FlashPfu DNA Polymerase 1 U/μl
  • 2× flashPfu PCR Buffer
  • 25 mM MgSO4

Description

TonkBio FlashPfu is a DNA polymerase for high-fidelity PCR. By fusing pfu-like polymerase with a processivity enhancing domain, we have dramatically increased it processivity and extension rate. FlashPfu DNA polymerase is about 50-fold more accurate than Taq polymerase and 6-fold more accurate than Pfu polymerase.

Applications

  • High Fidelity PCR
  • Fast PCR— extension 15-30 s/kb
  • Long range PCR —genomic DNA ≤ 19 kb, lambda DNA ≤ 30 kb)
  • Generation of PCR product for blunt-end cloning
  • Site-directed mutagenesis

Activity Determination

One unit of enzyme is defined as the amount of enzyme that will incorporate 10 nmol of dNTPs into acid insoluble material in 30 minutes at 75°C.

PCR Reaction Setup

Set up the following reaction in a sterile, nuclease-free microcentrifuge tube on ice.

Component Volume Final Concentration
2× FlashPfu PCR Buffer(with Mg2+) 25 μl
2.5 mM dNTP 4 μl 0.2 μM each
10 μM Forward Primer 2.5 μl 0.5μM (0.2−1.0 μM)
10 μM Reverse Primer 2.5 μl 0.5μM (0.2−1.0 μM)
Template DNA Variable Plasmid,Iambda,BAC DNA: 1−10 ng (1 pg−50 ng);
Genomic DNA: 100 ng (50−250 ng);
cDNA: 100 ng (50−500 ng)
FlashPfu DNA Polymerase 0.5-1.0 μl 0.5-1 U
Nuclease-free Water To 50 μl  
  • At 1× concentration, FlashPfu PCR Buffer provides 2.0 mM MgSO4. If optimization is desired, Mg2+ can be further optimized in 0.2 increments using MgSO4.
  • The addition of DMSO (final conc. 2–5%) is beneficial for the amplification of GC-rich targets. High DMSO concentrations (10%) will require lowering the annealing temperature by 5.5−6.0°C.

PCR Cycle Conditions

The Nearest Neighbor method is recommended to calculate the Tm of primers. The Tm values in this manual were calculated using this method with the following parameters:

  • Salt concentration: 80 mM;
  • Oligonucleotide concentration: 0.5 μM
  • Primer<25 mer or Tm<68°C----->3-step cycle
  • Primer≥25 mer or Tm≥68°C----->2-step cycle
3-Step Cycle
STEP TEMP. TIME CYCLES
Pre-denaturation 98°C 1-3 min 1
Denaturation 98°C 10 sec 25-35
Annealing (Tm+5) °C 20-30 sec
Extension 72°C 15−30 sec/kb
Final Extension 72°C 5-10 min 1
2-Step Cycle
STEP TEMP. TIME CYCLES
Pre-denaturation 98°C 1-3 min 1
Denaturation 98°C 10 sec 25-35
Extension 72°C 15−30 sec/kb
Final Extension 72°C 5-10 min 1
  • A temperature gradient can be used to optimize the annealing temperature for each primer pair.
  • Extension times are dependent on amplicon length and complexity. For low complexity DNA (e.g. plasmid, lambda, or BAC DNA) use 15 s/kb. For high complexity DNA (e.g. genomic DNA) use 30 s/kb. Do not exceed 1 min/kb.
  • Generally, 25–35 cycles result in optimal amplification of desired products. Up to 40 cycles may be required to detect low-copy-number targets.

Quality Control

Functional Assay: Performance of DNA amplification by PCR was confirmed by using human genomic DNA as the template (amplified fragments: 3.6 and 19 kb β-globin targets). The resulting PCR products are visualized as single bands on agarose gel.

  • Physical Purity: >95% pure by SDS-PAGE.

    Important Notes

    • Reactions using FlashPfu DNA polymerase yield best results when set up on ice prior to amplification. The polymerase has residual activity at temperatures above 4°C that can cause non-specific background amplification. It is important to add FlashPfu DNA Polymerase last, in order to prevent any primer degradation caused by the 3’→5’ exonuclease activity.
    • PCR products of FlashPfu DNA polymerase can be cloned according to blunt-end cloning methods.
    • Primer Design:
  • 1, Tm should be calculated using Nearest Neighbor Method.
  • 2, Primers should be 20–40 bases with Tm >60°C.
  • 3, Primers ideally have a GC content of 40–60%.
  • 4, If possible, the primer should terminate with a G or C at the 3’-end. But avoid multiple G or C bases at the 3’ end of the primer, because this may promote nonspecific binding.
  • 5, Primer melting temperatures should differ by no more than 5°C.
  • 6, The 3’ end of the primers should not be complementary to themselves or any other primer in the reaction.
  • 7, Primers for long target amplification (≥10 kb) should be 25-35 bases.
  • 8, Do not use uracil- or inosine-containing primers.

Datasheet Download

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