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Pfu-X Polymerase

Proofreading DNA polymerase for highest accuracy

Pyrococcus furiosus, recombinant, E. coli

Product Cat. No. Amount Price (EUR) Buy / Note
Pfu-X Polymerase PCR-207S 100 units 60,00 Add to Basket/Quote Add to Notepad
Pfu-X Polymerase PCR-207L 500 units 240,00 Add to Basket/Quote Add to Notepad

For in vitro use only!

Unit Definition: One unit is defined as the amount of the enzyme required to catalyze the incorporation of 10 nmol of dNTP into an acid-insoluble form in 30 minutes at 74 °C.

Shipping: shipped on blue ice

Storage Conditions: store at -20 °C
avoid freeze/thaw cycles

Shelf Life: 12 months

Form: liquid

Concentration: 2.5 units/μl

Related products:

  • Ready-to-Use Mixes / direct gel loading
  • Ready-to-Use Mixes
  • Thermophilic Polymerases
  • Deoxynucleotides (dNTPs)
  • Supplements
  • Primers and Oligonucleotides
  • DNA Ladders

Availability Restriction: Exclusively distributed in Japan by Greiner BioOne

Description:
Pfu-X Polymerase is the ideal choice for applications where the efficient amplification of DNA with highest fidelity is required. The enzyme is a genetically engineered Pfu DNA polymerase, but showing a 2-fold higher accuracy and an increased processivity, resulting in shorter elongation times.
The enzyme catalyzes the polymerization of nucleotides into duplex DNA in 5'→3' direction but does not possess a 5'→3' exonuclease replacement activity. Its inherent 3'→5' exonuclease proofreading activity results in a greatly increased fidelity of DNA synthesis compared to Taq polymerase. Pfu-X Polymerase-generated PCR fragments are blunt-ended. The enzyme is highly purified and free of bacterial DNA.

Fidelity of the enzyme:
Pfu-X Polymerase is characterized by a 50-fold higher fidelity compared to Taq polymerase and a 2-fold higher fidelity compared to standard Pfu polymerase.
ERPfu-X Polymerase = 0.25 x 10-6
The error rate (ER) of a PCR reaction is calculated using the equation ER = MF/(bp x d), where MF is the mutation frequency, bp is the number of base pairs of the fragment and d is the number of doublings
(2d = amount of product / amount of template).

Kit contents:
Pfu-X Pol (red cap)
2.5 units/μl Pfu-X Polymerase in storage buffer

Pfu-X Buffer (green cap)
10x conc.

Recommended 50 μl PCR assay:

5 μl10x Pfu-X Buffergreen cap
200 μMeach dNTP-
0.4 μMeach Primer-
1 - 100 ngtemplate DNA-
0.5 μl
(1.25 units)
Pfu-X Polred cap
Fill up to 50 μlPCR-grade water-
Please note that it is essential to add the polymerase as last component.
Recommended cycling conditions:
Three-step standard protocol
initial
denaturation
95 °C2 min1x
denaturation95 °C20 sec25-30x
annealing1)50 - 68 °C30 sec25-30x
elongation2)68 °C1 min/kb25-30x
final
elongation
68 °C1 min/kb1x

Two-step protocol for amplification of longer fragments (>3 kb)
Please note that for performing two-step cycling a sufficiently high primer Tm is necessary. If Tm of primers is below 65 °C or two-step PCR does not yield a sufficient product quality the three-step cycling protocol is recommended.
initial
denaturation
95 °C2 min1x
denaturation95 °C20 sec25-30x
annealing/
elongation1;2)
68 °C30 sec/kb25-30x
final
elongation
68 °C30 sec/kb1x

1) The annealing temperature depends on the melting temperature of the primers used.
2) The elongation time depends on the length of the fragments to be amplified. A time of 1 min/kb is recommended.

For optimal specificity and amplification an individual optimization of the recommended parameters may be necessary for each new template DNA and/or primer pair.