Cat.-no	Description	Amount	Price €	Shop
S127	Pfu/Psp RTL DNA Polymerase	1x250 units	65.00	add
S119	Pfu/Psp RTL DNA Polymerase	2x250 units	125.00	add
S120	Pfu/Psp RTL DNA Polymerase	10x250 units	570.00	add

Pfu/Psp red (RTL) DNA Polymerase: Order/request by E-mail:
Pfu/Psp red (RTL) DNA Polymerase: Datasheet                                                   RTL = ready-to-load 
Pfu/Psp red (RTL) 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. 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

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