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High Fidelity Hot Start Core Kit

Kit of heat-activatable DNA polymerase for high accuracy and specificity, dNTPs and reaction buffer

Product Cat. No. Amount Price (EUR) Buy / Note
High Fidelity Hot Start Core Kit PCR-235S 100 units 76,00 Add to Basket/Quote Add to Notepad
High Fidelity Hot Start Core Kit PCR-235L 500 units 304,00 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 Core Kit contains all reagents required for PCR (except template and primer) in one box combining simple handling with high flexibility. The premium quality polymerase, ultrapure dNTPs and the optimized complete reaction buffer ensure superior amplification results.
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.

Kit contents:

ComponentPCR-235SPCR-235L
High Fidelity Hot Start Pol
2.5 units/μl in storage buffer
red cap
40 μl
100 units
200 μl
500 units
dNTP Mix
10 mM each
dATP, dCTP,
dGTP, dTTP
white cap
100 μl500 μl
High Fidelity Buffer
10x conc.
green cap
500 μl2 x 1.2 ml

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).

Recommended 50 μl PCR assay:

5 μl10x High Fidelity Buffergreen cap
1 μldNTP Mixwhite cap
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.