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PFU MASTER MIX / LONG RANGE PCR MASTER MIX

ORDERING INFORMATION

HIGH FIDELITY PCR, 2x PFU MASTER MIX (pfu DNA Polymerase, 2.5 U/l)

Catalog #

Number of 50 l Reactions

Volume
5x Band Doctor

Price (EUR)

# R 550 100 2 x 1.25 ml 0.25 ml   

89,50

# R 551
500

10 x 1.25 ml

1.2 ml   
375,00

2x LONG RANGE PCR MASTER MIX (High Fidelity PCR Enzyme Mix, 2.5 U/l)

Catalog #

Number of 50 l Reactions

Volume
5x Band Doctor

Price (EUR)

# R 560 100 2 x 1.25 ml 0.25 ml   

89,50

# R 561
500

10 x 1.25 ml

1.2 ml   
375,00


DESCRIPTION

The 2x PCR Master Mix contains all reagents required for PCR and is designed to make PCR as easy and simple as possible. All components (including pfu DNA Polymerase and the High Fidelity PCR Enzyme Mix) are provided in an optimized concentration in the 2x PCR-Master solution. With 2x PCR Master Mix all you need to do is to add primers and template DNA, thus minimizing the pipetting effort and possible sources of error. The 2x pfu Master Mix is suitable for PCR amplification of DNA-fragments up to 4 kb. The 2x Long Range PCR Master Mix is suitable for PCR amplification of DNA-fragments up to 30 kb. In most cases even longer targets can be successfully amplified.


QUALITY CONTROL ASSAYS

FUNCTIONAL ASSAYS

SPECIFICATION

4 kb (High Fidelity PCR)

suitable

30 kb (Long and Accurate PCR)

suitable

PHYSICAL ASSAYS

SPECIFICATION

Endonuclease Assay

no activity detected

DNase Assay

no activity detected

RNase Assay

no activity detected


BASIC PROTOCOL

Mix the following components on ice in a PCR-tube and adjust to a final volume of 50 l with water:

Component

Volume (in l)

Mix 1

Mix 2

Mix 3

Mix 4

Mix 5

2x PCR Master Mix

25

25

25

25

25

Template DNA

x

x

x

x

x

Primer (10 M, each)

2

2

2

2

2

5x Band Doctor

0

5

10

15

20

2x distilled., sterile water

add up to 50

add up to 50

add up to 50

add up to 50

add up to 50

Mix gently and place in a thermal cycler. No vortexing, no centrifugation! Optimal conditions for concentrations of primer, template and temperature profile need to be determined for each reaction.


CYCLING PROGRAM FOR 4 kb PCR ASSAY

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

2 min/kb

10 40

Final Extension

72

5 min

1

CYCLING PROGRAM FOR 30 kb PCR ASSAY

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

30 sec/kb

10 40

Final Extension

72

5 min

1

AT* : Annealing Temperature; Choose to 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. 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.

C. 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.

D. Extension Time

The extension time should be 2 min/kb for pfu Master Mix. For Long Range PCR Master Mix 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 12 months.