Taq Pol Hot Start Ab+ dual buffer | Taq DNA Polimerase Hot Start Cellco

Taq Pol Hot Start Ab+ dual buffer

Ficha técnica

Código	Taq Pol Hot Start Ab+ dual buffer
Categoria	PCR Convencional
Marca	Cellco

Descrição Geral

Taq Pol Hot Start Ab+ dual buffer | Taq DNA Polimerase Hot Start - Cellco
DNA polimerase ativada por temperatura, bloqueio por anticorpo, dual buffer
Thermus aquaticus, 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: 12 months

Concentração: 5 units/μL

Kit contents:

Taq Pol Hot Start (blue cap)

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

Taq HS Reaction Buffer (red cap) - 5x conc.

Proprietary formulation supplied at pH 8,5 (25°C). Does not contain dyes or magnesium.

Taq HS Reaction Buffer Green (red cap) - 5x conc.

Is supplemented with tracking dyes for direct loading of PCR products on gels. Does not contain magnesium.

MgCl Stock Solution (yellow cap)

50 mM MgCl₂.

Descrição:

Hot Start Pol provides improved specificity and sensitivity when amplifying low-copy-number targets in complex backgrounds or when prolonged room-temperature set up is required. The polymerase activity is blocked at ambient temperature and switched on automatically at the onset of the initial denaturation. The thermal activation prevents the extension of nonspecifically annealed primers and primer-dimer formation at low temperatures during PCR setup. The enzyme catalyzes the polymerization of nucleotides into duplex DNA in 5’→3’ direction in the presence of magnesium. It also possesses a 5’→3’ polymerization-dependent exonuclease replacement activity but lacks a 3’→5’ exonuclease activity.

Activation step:

Hot Start Pol requires no prolonged heating or denaturing step. The polymerase inhibiting antibody is quickly released at the increased temperature of thermal cycling.

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	-
5x Reaction Buffer	10 μl	1x
dNTP Mix 10 mM	1 μl	200 μM
Taq Pol Hot Start (5U/ µL)	0,25-0,5 μl	1,25 - 2,5 U/reaction
MgCl₂	2 µl	2 mM

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. Optimization of MgCl₂ concentration:

The 5x reaction buffer does not contain MgCl₂. For an individual 2 optimization, add MgCl₂ stock solution as shown in the table 2 below:

MgCl₂ Final Concentration	2,5 mM	3 mM	4 mM
MgCl₂ Stock volume to 50 μl	2,5 μl	3 μl	4 μl

4. Incubate reactions in a thermal cycler

Recommended cycling conditions:

Initial denaturation	95 °C	2 – 5 min	1x
Denaturation	95 °C	15 - 30 sec	30 cycles
Annealing**	45 - 68 °C	15 - 30 sec	30 cycles
Elongation***	72 °C	1 min/kb	30 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.

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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