Pfu/Psp red (RTL) DNA Polymerase: Datasheet
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. For visual control and for fast loading on the gel the enzyme contains a red dye and loading buffer for agarose electrophoresis.
Applications:
- blunt end PCR cloning
- PCR and primer extension where "high fidelity" is required
- Site-directed mutagenesis
- PCR where visual control is needed
Description:
Pfu/Psp DNA polymerase ready-to-load is isolated from the archae bacteria Pyrococcus f-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/Psp DNA Polymerase has no detectable reverse transcriptase activity.
Concentration: (1 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 and red dye
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 (1 u/µl) |
1 - 2 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 1 kb)
|
95°C
37-69°C
72-75°C
|
|
Final extension |
5 min |
72-75°C |
Loading on the gel:
Recommended volume is 10 µl of reaction mixture
Storage: at -20°C for 24 months
Transportation: on blue ice
|
