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Hot Start Polymerase Ab+ - glycerol-free

Heat-activatable DNA polymerase for high specificity, antibody-blocked in glycerol-free storage buffer

Thermus aquaticus, recombinant, E. coli

Cat. No. Amount Price (EUR) Buy / Note
PCR-424-1KU 1 kilo unit 450,80 Add to Basket/Quote Add to Notepad
PCR-424-10KU 10 kilo units 2.254,00 Add to Basket/Quote Add to Notepad

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

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.

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.

componentfinal assay conc.
Reaction Buffer1 x
dNTP Mix200 μM
Hot Start Polymerase Ab+0.025-0.05 units/μl
primer mix or each primer200-400 nM each primer
PCR-grade Waterfill up to final volume

Cycling Conditions:

95 °C2 min1 x
annealing 1)
elongation 2)
95 °C
50-68 °C
72 °C
10-20 sec
10-20 sec
20 sec - 4 min
25-30 x

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.

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