HIGH FIDELITY DNA POLYMERASES

ORDERING INFORMATION

PFU DNA POLYMERASE (2.5 U/µl)

Catalog #	Units	10x pfu Reaction Buffer	5x Band Doctor	Price (EUR)
# PF 100	100	0.5 ml	0.25 ml	49,00
# PF 500	500	2.4 ml	1.2 ml	200,00

LONG HIGH FIDELITY PCR ENZYME MIX (2.5 U/µl)

Catalog #	Units	10x HF  Reaction Buffer	5x Band Doctor	Price (EUR)
# HF 100	100	0.5 ml	0.25 ml	49,00
# HF 500	500	2.4 ml	1.2 ml	200,00

DESCRIPTION

Pfu DNA Polymerase is a highly purified recombinant highly thermostable DNA polymerase that has been isolated from E. coli carrying a vector encoding the Pyrococcus furiosus DNA polymerase gene. The enzyme is twenty-five times more accurate than Taq DNA Polymerase and it produces blunt-end amplicons.

High Fidelity DNA Polymerase is a blend of DNA polymerases specially designed for the highly accurate and efficient amplification of fragments up to 30 kb including GC-rich or other difficult templates.

Unit Definition

Storage buffer pfu Polymerase

50 mM Tris/HCl (pH 8.0 bei 25°C), 1 mM DTT, 0.1 mM EDTA, 0.1% (v/v) Nonidet P-40, 1 mM PMSF, 0.1% (v/v) Tween 20, 50% (v/v) Glycerol.

Storage buffer HF Polymerase

20 mM Tris/HCl (pH 8.0 bei 25°C), 100 mM KCL, 1 mM DTT, 0.1 mM EDTA, 0.1% (v/v) Nonidet P-40, 1 mM PMSF, 0.1% (v/v) Tween 20, 50% (v/v) Glycerol.

10x Reaction Buffer

Endonuclease Assay

Exonuclease Assay

Ribonuclease Assay

Functional Assay

BASIC PROTOCOL

Component	Volume (in µL)
10x pfu/HF Reaction Buffer	5
10 mM dNTP-Mastermix	1
Forward-Primer (10 pmol/µl)	2
Reverse-Primer (10 pmol/µl)	2
Template DNA	variable
5x Band Doctor	0 – 20
pfu/HF Polymerase (2.5 U/µl)	0.5
2x distilled, sterile water	Add to a final volume of  50
Total volume	50

CYCLING PROGRAM

Step	Temperature (in °C)	Time	Cycles
Initial activation	95	2 min	1
Denaturation	95	20 sec	10 – 40
Annealing	AT*	40 sec	10 – 40
Extension	72	See chapter E (Application)	10 – 40
Final Extension	72	5 min	1

AT* : Annealing Temperature; Choose the lower T_m of  both primers;

AT = T_m – 5°C ; T_m = 2°C x (A+T) + 4°C x (G+C)

APPLICATION

A. Template

Template DNA	DNA Amount	Number of Cycle
Animal genomic DNA	50 – 200 ng	25 – 35
	10 – 50 ng	30 – 40
Bacterial genomic DNA	10 – 50 ng	20 – 25
	1 – 5 ng	30 – 35
Plasmid and Lambda DNA	1 – 5 ng	20 – 30

B. DNA Polymerase

For amplification of longer fragments of an animal genomic DNA, the amount of enzyme should be increased to 2 or 2.5 U.

C. 5x Band Doctor

The use of Band doctor is not necessary in general, but helps in the amplification of DNA with high GC content and complex structures. The included 5x Band doctor can be added to the reaction mixture at the final concentration of 0.5x – 2x.

Reaction mixture (conc. of band doctor)

Component	Volume (in µl)
	Mix 1	Mix 2	Mix 3	Mix 4	Mix 5
10x pfu/HF Reaction Buffer	5	5	5	5	5
10 mM dNTP-Mastermix	1	1	1	1	1
Forward-Primer (10 pmol/µl)	2	2	2	2	2
Reverse-Primer (10 pmol/µl)	2	2	2	2	2
Template DNA	x	x	x	x	x
5x Band Doctor	0	5	10	15	20
pfu/HF Polymerase (2.5 U/µl)	0.5	0.5	0.5	0.5	0.5
2x distilled, sterile water	up to 50	up to 50	up to 50	up to 50	up to 50

D. 5x Primer Design

E. Extension Time

 

STORAGE

Store at -20°C for 24 months.