Heat-activatable DNA polymerase for high specificity, chemically modified
Thermus aquaticus, recombinant, E. coli
| Cat. No. | Amount | Price (EUR) | Buy / Note |
|---|---|---|---|
| PCR-218S | 200 units | 89,80 | Add to Basket/Quote Add to Notepad |
| PCR-218L | 1.000 units | 359,00 | 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 nmoles of dNTP's into an acid-insoluble form in 30 minutes at 70 °C.
Shipping: shipped on blue ice
Storage Conditions: store at -20 °C
avoid freeze/thaw cycles
Shelf Life: 12 months
Form: liquid
Concentration: 5 units/μl
Description:
Hot Start KlenTaq provides an improved specificity and sensitivity when amplifying low-copy-number targets in complex backgrounds or when room-temperature set up is required. The polymerase activity is chemically blocked at ambient temperature preventing the extension of nonspecifically annealed primers and primer-dimer formation at ambient temperature during PCR setup. The enzyme catalyzes the polymerization of nucleotides into duplex DNA in 5’→3’ direction but lacks the 5’→3’ exonuclease activity of Taq polymerase. The enzyme is purified by an additional separation process to reduce contaminating bacterial DNA sequences.
In addition to routine PCR, Hot Start KlenTaq is particularly recommended for genotyping applications.
Activation step
Hot Start KlenTaq requires an initial denaturing step of 10-12 min to release the chemical modification of the polymerase.
Content:
Hot Start KlenTaq (red cap)
5 units/μl Hot Start KlenTaq DNA Polymerase in 50 mM Tris-HCl, 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5 % Tween-20, 0.5 % Nonidet P-40, 50 % (v/v) Glycerol, pH 9.0 (25°C)
Hot Start KlenTaq Buffer (green cap), 10x conc.
100 mM Tris-HCl, 160 mM (NH4)2SO4, 35 mM MgCl2, 0.25 % Brij58, 0.5 % NP40, 2 mg/ml BSA, pH 8.5 (25 °C)
| component | PCR-218S | PCR-218L |
| Hot Start KlenTaq Polymerase | 200 units / 40 μl | 1000 units / 200 μl |
| Hot Start KlenTaq Buffer, 10x conc. | 400 μl | 5 x 400 μl |
PCR Reaction Set-Up:
The PCR procedure below shows appropriate volumes for a single 50 μl reaction. For multiple reactions, prepare a master mix of all components and dispense appropriate volumes into each PCR reaction tube prior to adding template DNA and primers.
Thaw up and briefly centrifuge each component before use.
Add the following components to a PCR tube:
Prepare PCR Master Mix
| comp. | cap | final conc. | 1 assay @ 50 μl |
| PCR-grade Water | white | fill up to 50 μl | |
| 10x reaction Buffer | green | 1x | 5 μl |
| dNTP Mix 10 mM | white | 200 μM each | 1 μl |
| KlenTaq | red | 1.25 - 2.5 units/reaction | 0.25 - 0.5 μl |
| forward primer (10 μM) | 0.1 - 0.5 μM | 0.5 - 2.5 μl | |
| reverse primer (10 μM) | 0.1 - 0.5 μM | 0.5 - 2.5 μl | |
| template DNA | 10 pg - 1 μg1) |
Mix and briefly centrifuge the components
Optimisation of MgCl2 concentration:
The final assay contains 3.5 mM Mg2+ as recommended for most applications. For an individual optimisation Mg2+ stock solution (#PCR-266) mybe added.
Incubate reactions in a thermal cycler
Recommended cycling conditions:
| initial denaturation | 95 °C | 10 - 12 min | 1x |
| denaturation annealing2) elongation3) | 95 °C 50 - 68 °C 72 °C | 15 - 30 sec 15 - 30 sec 30 sec - 4 min | 25 - 35x |
For optimal specificity and amplification an individual optimisation of the recommended parameters may be necessary for each new template DNA and/or primer pair.
Related products: Mg2+ Stock, #PCR-266
