Taq Pol Hot Start Ab+ (sem MgCl2) | Taq DNA Polimerase Hot Start Cellco

Taq Pol Hot Start Ab+ (sem MgCl2)

Ficha técnica

Código	Taq Pol Hot Start Ab+ (sem MgCl2)
Categoria	PCR Convencional
Marca	Cellco

Descrição Geral

Taq Pol Hot Start Ab+ (sem MgCl₂) | Taq DNA Polimerase Hot Start - Cellco
DNA polimerase ativada por temperatura, bloqueio por anticorpo
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 - POL-123 (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 Reaction Buffer (-) Mg+² (red cap) - 10x conc.

Tris-HCl pH 8.5 (25°C) and KCl.

MgCl2 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. The engineered Taq DNA Pol hot start enzyme allows amplification of fragments up to 5 kbp.

Activation step:

Hot Start Pol does not require a prolonged heating or denaturing step. The polymerase inhibiting antibody is quickly released at the increased temperature of the hot start cycle.

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 DNA Polymerase (5 U/μl)	0,25-0,5 μl	1,25-2,5 U/assay
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:

For an individual optimization add MgCl₂ stock solution as shown 2 in the table below.

MgCl₂ Final Concentration	2,5 mM	3 mM	4 mM
MgCl₂Stock 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/kbp	30 cycles
Final extension (optional)	72 °C	1 – 2 min/kbp	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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