ORDERING INFORMATION

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

Catalog #	Number of 50 µl 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/µl)

Catalog #	Number of 50 µl 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 µl with water:

Component	Volume (in µl)
	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 µM, 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 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. 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

D. Extension Time

STORAGE

Store at -20°C for 12 months.