Hot Start Polymerase Ab⁺ - glycerol-free

For general laboratory use.

Unit Definition: One unit is defined as the amount of the enzyme required to catalyze the incorporation of 10 nmoles of dNTP's into an acid-insoluble form in 30 minutes at 70 °C using hering sperm DNA as substrate.

Shipping: shipped on gel packs

Storage Conditions: store at -20 °C
avoid freeze/thaw cycles

Shelf Life: 6 months

Form: liquid

Concentration: 5 units/μl

Description:
Hot Start Polymerase Ab⁺ - glycerol-free is recommended for use in freeze drying applications where glycerol must be avoided.
The polymerase 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 initial denaturation. The thermal activation prevents the extension of non-specifically annealed primers and primer-dimer formation at low temperatures during PCR setup. The polymerase is recommended for diagnostic applications, high throughput PCR or genotyping.
The enzyme replicates DNA at 72 °C. It 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 (proof-reading) activity.

Activation step
Hot Start Polymerase Ab⁺ requires no prolonged heating or denaturing step. The polymerase inhibiting antibody is released within 2 min at 92°C during the initial denaturation step.

Content:
Reaction Buffer and Nucleotides are not included. Please refer to our our sections Buffers & Components and Nucleotides.

Hot Start Polymerase Ab⁺
5 units/μl Hot Start Polymerase Ab⁺ in 20 mM Tris-HCl, 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, stabilizer, cryo-protector, pH 9.0

Preparation of the master mix:
Before starting, thaw up all components and vortex thoroughly to ensure homogeneity.

Cycling Conditions:

¹⁾ The annealing temperature depends on the melting temperature of the primers used.
²⁾ The elongation time depends on the length of the fragments to be amplified. A time of 1 min/kb is recommended.

Recommended Buffer Systems:
qPCR Buffer (#PCR-279) is recommended for use in real-time PCR applications. The buffer contains a well-balanced ratio of potassium-, ammonium- and magnesium-ions to ensure high specificity and minimal by-product formation in probe-based assays as well as in SybrGreen/EvaGreen-based assays. There is no need of additional optimization.,

KCl Buffer (#PCR-262) is recommended for use in routine PCR reactions. The buffer is optimized for highest specificity but may require additional fine-tuning of assay parameters like MgCl₂ concentration and annealing temperature.

Optimization of MgCl₂ concentration:
MgCl₂ Solution - 25 mM (#PCR-266) is recommended for optimization of the final Mg²⁺ concentration. A concentration of 1.5-2.0 mM is required for optimal functionality. Lower concentrations give higher specificity, whereas higher concentrations give higher yield.

Related products:

• dNTP Mix - 25 mM, #NU-1023
• qPCR Buffer, #PCR-279
• Crystal Buffer, #PCR-271
• KCl Buffer, #PCR-262
• MgCl₂ Solution - 25 mM, #PCR-266"