High Fidelity Hot Start Pol

Heat-activatable DNA polymerase for high accuracy and specificity

Thermus species, recombinant, E. coli

Product Cat. No. Amount Price (EUR) Buy / Note
S pack PCR-205S 100 units 68,00 Add to Basket/Quote Add to Notepad
L pack PCR-205L 500 units 272,00 Add to Basket/Quote Add to Notepad

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

Form: liquid

Concentration: 2.5 units/μl

Related products:

  • Ready-to-Use Mixes / direct gel loading
  • Ready-to-Use Mixes
  • Thermophilic Polymerases
  • Deoxynucleotides (dNTPs)
  • Supplements
  • Primers and Oligonucleotides
  • DNA Ladders

Availability Restriction: Exclusively distributed in Japan by Greiner BioOne

Description:
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.
Activation step:
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).
Kit contents:
High Fidelity Hot Start Pol (red cap)
2.5 units/μl High Fidelity Hot Start Polymerase in storage buffer
High Fidelity Buffer (green cap)
10x conc.

Recommended 50 μl PCR assay:

5 μl10x High Fidelity Buffergreen cap
200 μMeach dNTP-
0.2 - 0.5 μMeach Primer-
1 - 100 ngtemplate DNA-
0.5 μl
(1.25 units)
High Fidelity Hot Start Polred cap
Fill up to 50 μlPCR-grade water-

Please note that it is essential to add the polymerase as last component.

Recommended cycling conditions:

initial
denaturation
95 °C2 min1x
denaturation95 °C20 sec20-30x
annealing1)50 - 68 °C30 sec20-30x
elongation2)68 °C1 min/kb20-30x
final
elongation
68 °C1 min/kb1x

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.

For optimal specificity and amplification an individual optimization of the recommended parameters may be necessary for each new template DNA and/or primer pair.