Pfu-X Polymerase

For general laboratory use.

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 gel packs

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

Shelf Life: 12 months

Form: liquid

Concentration: 2.5 units/μl

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.
ER_{Pfu-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
(2^d = amount of product / amount of template).

Content:
Pfu-X Pol (red cap)
2.5 units/μl Pfu-X Polymerase in storage buffer
(50 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 1 mM DTT, stabilizers, 50% Glycerol)

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

Recommended 50 μl PCR assay:

Please note that it is essential to add the polymerase as last component.

Recommended cycling conditions:
Three-step standard protocol

Two-step protocol for amplification of longer fragments (>3 kb)
Please note that for performing two-step cycling a sufficiently high primer T_m is necessary. If T_m of primers is below 65 °C or two-step PCR does not yield a sufficient product quality the three-step cycling protocol is recommended.
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.

Related products:

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

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