Description:
Pfu DNA Polymerase is a thermostable
enzyme of approximately 90kDa isolated from Pyrococcus
furiosus. The enzyme replicates DNA at 75oC,
catalyzing the polymerization of nucleotides into duplex
DNA in the 5´-3´ direction in the presence
of magnesium. Pfu DNA Polymerase also possesses 3´-5´
exonuclease (proofreading) activity. Base misinsertions
that may occur during polymerization are rapidly excised
by the proofreading activity of the polymerase.
Consequently, Pfu DNA Polymerase is recommended for use
in PCR and primer extension reactions that require
high-fidelity synthesis. Pfu DNA Polymerase-generated
PCR fragments are blunt-ended.
Concentration:5U/ul
Source:
Pyrococcus furiosus
strain Vc1 DSM3638. 10X Reaction Buffer with MgSO4:
200mM
Tris-HCl (pH 8.8 at 25oC),
100mM
KCl,
100mM
(NH4)2SO4,
20mM
MgSO4, 1.0% Triton X-100 and 1mg/ml
nuclease-free BSA.
Features:
High Fidelity: Pfu DNA Polymerase
exhibits the lowest error rate of any thermostable DNA
polymerase. Applications:
Pfu DNA Polymerase is recommended for
use in PCR, primer extension reactions and other
applications that demand high fidelity.
Unit
Definition:
One unit is defined as the amount of
enzyme required to catalyze the incorporation of 10nmol
of dNTPs into acid-insoluble material in 30 minutes at
75oC.
Reaction
Mixture Set Up
1.
Gently vortex and briefly centrifuge all solutions after
thawing.
2.
Keep solutions on ice.
3.
Add to a thin-walled PCR tube, on ice:
Reagent
Quantity,
for 50 μl of reaction mixture
Final
concentration
10X
Pfu buffer
5ul
1X
10mM
dNTP
1ul
0.2mM
PrimerI(25pmol/ul)
1ul
0.5pmol/ul
PrimerII(25pmol/ul)
1ul
0.5pmol/ul
Pfu(5U/ul)
0.4ul
2U/50ul
Template
DNA
variable
50
pg -1 μg
ddH2O
Up
to 50ul
4.
Gently vortex the sample and briefly centrifuge to
collect all drops from walls of tube.
5. If
using a thermal cycler without a heated lid, overlay the
sample with a half volume of mineral
oil
or add an appropriate amount of wax.
6.
Place samples in a thermal cycler preheated to
96°C
and start PCR.
Recommended
thermal cycling conditions:
Step
Temperature,°C
Time ,min
Number
of cycles
Initial
denaturation
96
4
1
Denaturation
95
0.5
Annealing
50-68
0.5-2
25-35
Extension
72
0.5-4
Final
Extension
72
5
1
Quality
Control Tests:
PCR (activity), SDS-PAGE (purity), endonuclease/nickase.
Storage:
Pfu DNA Polymerase in
50mM
Tris-HCl (pH 8.2 at 25oC),
0.1mM
EDTA,
1mM
DTT, 50% glycerol and 0.05% CHAPS should be stored at
-20oC.Use
dry ice for shipping.
