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HIGH FIDELITY DNA POLYMERASES

ORDERING INFORMATION

PFU DNA POLYMERASE (2.5 U/l)

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/l)

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

One unit catalyzes the incorporation of 10 nmol of deoxyribonucleotides into a polynucleotide fraction (adsorbed on DE-81) in 30 minutes at 72C.

Storage buffer pfu Polymerase

50 mM Tris/HCl (pH 8.0 bei 25C), 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 25C), 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

500 mM Tris/HCl, 140 mM (NH4)2SO4, 17.5 mM MgCl2 (pH 9.1 at 25C).

Endonuclease Assay

No detectable conversion of covalently closed circular DNA to nicked DNA was observed after incubation of 15 units of pfu/High Fidelity DNA Polymerase with 0.5 g of pUC-19-plasmid-DNA in 10 l of 1x Reaction Buffer containing 1.5 mM MgCl2 for 2 hours at 72C.

Exonuclease Assay

No detectable degradation (smearing) of fragments was observed after incubation of 5 units of pfu/High Fidelity DNA Polymerase with 0.5 g of pUC-19-plasmid-DNA (digested with Hpa II) in 10 l of 1x Reaction Buffer containing 1.5 mM MgCl2 for 2 hours at 72C.

Ribonuclease Assay

No detectable degradation of 28S/18S bands was observed after incubation of 5 units of pfu/High Fidelity DNA Polymerase with 1 g of total RNA (from rat liver) in 10 l of 1x Reaction Buffer containing 1.5 mM MgCl2 for 2 hours at 72C.

Functional Assay

pfu DNA Polymerase and High Fidelity DNA Polymerase were tested for amplification of a 20 kb fragment of human genomic DNA.


BASIC PROTOCOL

Component

Volume (in L)

10x pfu/HF Reaction Buffer

5

10 mM dNTP-Mastermix

1

Forward-Primer (10 pmol/l)

2

Reverse-Primer (10 pmol/l)

2

Template DNA

variable

5x Band Doctor

0 20

pfu/HF Polymerase (2.5 U/l)

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 Tm of  both primers;

AT = Tm 5C ; Tm = 2C x (A+T) + 4C 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 l)

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/l)

2

2

2

2

2

Reverse-Primer (10 pmol/l)

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/l)

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

Primers can be designed manually or using a primer design software. It is recommended that Tm of the designed primers is above 64C, and AT above 58C.

E. Extension Time

 

The extension time should be 2 min/kb for pfu Polymerase. For High Fidelity Polymerase the extension time should be 30 sec/kb for short fragments and 1 min/kb for fragments longer than 5 kb.

For the amplification of fragments longer than 5 kb, the temperature ought to be assigned at 68C.


STORAGE

Store at -20C for 24 months.