Pfu/Psp DNA Polymerase: Datasheet
Pfu/Psp DNA Polymerase: Deutsche Beschreibung
Features:
Pfu/Psp DNA polymerase replicates DNA at 75°C catalyzing the polymerization of nucleotides into duplex DNA in the 5´=>3´ direction in the presence of Mg+. Pfu DNA polymerase possesses 3' to 5' exonuclease proof reading activity that enables the polymerase to correct nucleotide-misincorporation errors. The enzyme has no 5'=>3' exonuclease activity.
Applications:
- blunt end PCR cloning
- PCR and primer extension where "high fidelity" is required
- Site-directed mutagenesis
Description:
Pfu/Psp DNA polymerase, isolated from the archae bacteria Pyrococcus furiosus/species is a thermostable Polymerase of approximately 90000 daltons. Base misinsertions that may occur during polymerization are rapidly excised by the proofreading activity of the polymerase. The Pfu DNA Polymerase has no detectable reverse transcriptase activity.
Concentration: 5 u/µl
Unit definition:
One unit is defined as the amount of enzyme required to catalyze the incorporation of 10 nM of dNTPs into acid insoluble material in 30 minutes at 75°C.
Storage Buffer:
50 mM Tris-HCl, pH 8.2, 0.1 mM EDTA, 0.1% Tween 20, 0.1% Nonident P40, 1 mM DTT, 50% Glycerol
Reaction Buffer 10 X:
100 mM KCl, 160 mM (NH4)2SO4, 20 mM MgSO4, 200 mM Tris-HCl, pH8.8, 1% Triton X-100, 1 mg/ml BSA
Quality control:
- Tested for the DNA amplification of 2,2 kb from lambda DNA
- Contamination level check of bacterial DNA
- Purity by SDS-Page > 90 %
Usage:
Standard protocol:
- Do not use dUTP or dITP or primers containing these nucleotides
|
Components |
Volume per reaction |
end conc. |
|
10X reaction buffer with MgSO4 |
5 µl |
1X |
|
dNTP-Mix (40mM = 10mM each) |
1.0 µl |
200 µM each |
|
Up-stream primer (e.g. 20 µM) |
0,5 µl |
0.1-1.0 µM |
|
Down-stream primer (e.g. 20 µM) |
0.5 µl |
0.1-1.0 µM |
|
Template DNA (10 ng/µl) |
1.0 µl |
<= 0,5 µg |
|
Pfu/Psp DNA Polymerase (5 u/µl) |
0.2 - 0,4 µl |
1-2 units |
|
Sterile dest. Water (molecular grade) |
up to 50 µl
|
|
Note:
- vortex all solutions carefully before using
- dispense all reagents on ice to avoid degradation of primers and dNTP's
- add the enzyme after Template DNA
- may you have to optimize the MgSO4 concentration for best result
General Thermo-Cycler protocol:
|
Step |
Time |
Temperature |
|
Initial denaturation |
1-3 min |
95°C |
25-35 Cycles:
Denaturation
Annealing
Extension
|
30-100 sec
30-65 sec
1-2 min (per 1kb) |
95°C
37-69°C
72-75°C
|
|
Final extension |
5 min |
72-75°C |
Storage: at -20°C for 24 months
Transportation: on blue ice
|
