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

 

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

 

Tris-HCl pH 8.5 (25°C), KCl, 20 mM MgCl₂.

 

25 mM MgCl₂.

 

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.

 

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

 

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:

 

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

Mix and briefly centrifuge the components.

 

 

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

 

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

 

The 10x reaction buffer contain 20 mM MgCl₂, a recommended concentration for most applications. For an individual optimization add MgCl₂ stock solution as shown in the table below.

 

Recommended cycling conditions:

**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.