Heat-activatable DNA polymerase for high accuracy and specificity
Thermus species, recombinant, E. coli
For in vitro use only!
Unit Definition: One unit is defined as the amount of the enzyme required to catalyze the incorporation of 10 nmol of dNTP into an acid-insoluble form in 30 minutes at 74 °C.
Shipping: shipped on blue ice
Storage Conditions: store at -20 °C
avoid freeze/thaw cycles
Shelf Life: 12 months
Concentration: 2.5 units/μl
High Fidelity Hot Start Pol is based on a blend of Taq DNA polymerase and a proofreading enzyme specially designed for highly accurate and efficient amplification. The additional hot-start function 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 shows excellent results with extremely long (up to 30 kb), GC-rich or other difficult templates. The enzyme blend includes a highly processive 5'→3' DNA polymerase and possesses a 5'→3' polymerization-dependent exonuclease replacement activity. Its inherent 3'→5' exonuclease proofreading activity results in a greatly increased fidelity of DNA synthesis compared to Taq polymerase. The enzyme is highly purified and free of bacterial DNA.
High Fidelity Hot Start Pol requires no prolonged heating or denaturing step. The polymerase inhibiting antibodies are released at the increased temperature of the initial denaturation.
Fidelity of the enzyme:
High Fidelity Pol is characterized by a 4-fold higher fidelity compared to Taq polymerase.
ERHigh Fidelity Pol = 3.4 x 10-6
The error rate (ER) of a PCR reaction is calculated using the equation ER = MF/(bp x d), where MF is the mutation frequency, bp is the number of base pairs of the fragment and d is the number of doublings
(2d = amount of product / amount of template).
High Fidelity Hot Start Pol (red cap)
2.5 units/μl High Fidelity Hot Start Polymerase in storage buffer
High Fidelity Buffer (green cap)
Recommended 50 μl PCR assay:
|5 μl||10x High Fidelity Buffer||green cap|
|200 μM||each dNTP||-|
|0.2 - 0.5 μM||each Primer||-|
|1 - 100 ng||template DNA||-|
|High Fidelity Hot Start Pol||red cap|
|Fill up to 50 μl||PCR-grade water||-|
Recommended cycling conditions:
|95 °C||2 min||1x|
|denaturation||95 °C||20 sec||20-30x|
|annealing1)||50 - 68 °C||30 sec||20-30x|
|elongation2)||68 °C||1 min/kb||20-30x|
|68 °C||1 min/kb||1x|
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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