Taq High Fidelity Polimerase | Taq DNA Polimerase High Fidelity Cellco

Taq High Fidelity Polimerase

Ficha técnica

Código	Taq High Fidelity Polimerase
Categoria	PCR Convencional
Marca	Cellco

Descrição Geral

Taq High Fidelity Polimerase | Taq DNA Polimerase High Fidelity - Cellco
DNA polimerase de elevada fidelidade
Thermus sp, recombinante, E. coli

For in vitro use only!

Definição de unidade: One unit is defined as the amount of the enzyme required to catalyze the incorporation of 10 nmoles of dNTPs into an acidinsoluble form in 30 minutes at 70 °C.

Envio: Shipped on blue ice

Condições de armazenamento: Store at -20 °C

Avoid freeze/thaw cycles

Validade: 36 months

Concentração: 5 units/μL

Kit contents:

Taq High Fidelity Pol (blue cap)

5 units/µl Taq High Fidelity Pol in Tris-HCl, KCl, EDTA, DTT, 50% (v/v) Glycerol, pH 8.0 (25°C) and stabilizers.

Taq High Fidelity Pol Reaction Buffer complete (red cap) - 10x

Tris-HCl pH 9,0 (25°C), KCl, 15 mM MgCl₂.

Descrição:

Taq High Fidelity Pol is based on a blend of Taq DNA polymerase and a proofreading enzyme specially designed for highly accurate and efficient amplification. It shows excellent results with extremely long (up to 25 kb), GC-rich or other difficult templates. The enzyme blend includes a highly processive 5’→3’ DNA polymerase and possesses a 5’3’ polymerizationdependent exonuclease replacement activity. Its inherent 3’→5’ exonuclease proofreading activity results in a greatly increased fidelity of DNA synthesis compared to Taq polymerase. The enzyme is highly purified and free of bacterial DNA.

Fidelidade da Enzima:

Taq High Fidelity Pol is characterized by a 2-fold higher fidelity compared to Taq polymerase.

PCR Reaction Setup:

The PCR procedure below shows appropriate volumes for a single 50-μL reaction. For multiple reactions, prepare a master mix of components common to all and then dispense appropriate volumes into each PCR reaction tube prior to adding template DNA and primers. Thaw, mix, and briefly centrifuge each component before use.

Add the following components to a microcentrifuge tube:

1. Prepare PCR master mix

Note: Consider the volumes for all components listed next steps to determine the correct amount of water required to reach your final reaction volume.

Component	50 μL rxn	Final conc.
Water, grade PCR	to 50 μl	-
10x Reaction Buffer	5 μl	1x
dNTP (Mix 10 mM)	1 μl	200 μM
Taq High fidelity DNA Pol (5U/ µl)	0,25 - 0,5 μl	1,25 - 2,5 U/reaction

Mix and briefly centrifuge the components.

2. Add template DNA and primers

Component	50 μl rxn	final conc.
Forward primer (10 μM)	0,5 – 2,5 μl	0,1 – 0,5 μM
Reverse primer (10 μM)	0,5 – 2,5 μl	0,1 – 0,5 μM
Template DNA	x μl	10 pg – 1 μg*

*genomic DNA: 1 ng – 1 μg; plasmidial DNA: 1 pg – 1 ng

Cap each tube, mix, and briefly centrifuge the content.

3. Incubate reactions in a thermal cycler

Recommended cycling conditions:

Initial denaturation	95 °C	1 - 3 min	1x
Denaturation	95 °C	15 - 30 sec	25-35 cycles
Annealing**	45 - 70 °C	15 - 30 sec	25-35 cycles
Elongation***	72 °C	1 min/kb	25-35 cycles
Final extension (optional)	72 °C	1 – 2 min/kb	1x
Hold	4 – 8 °C	∞	1x

**The annealing temperature depends on the melting temperature of the primers used

***The elongation time depends on the length of the fragments to be amplified. A time of 1 min/kb is recommended.

Note: For large fragments it is recommended in the elongation step to use 68 ºC and 1,5 - 2 min/kbp.

For optimal specificity and amplification an individual optimization of the recommended parameters may be necessary for each new template DNA and/or primer pair.

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