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Hot Start KlenTaq

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 87,74 Add to Basket/Quote Add to Notepad
PCR-218L 1.000 units 350,55 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)

componentPCR-217SPCR-217L
Hot Start KlenTaq
Polymerase
200 units
/ 40 μl
1000 units
/ 200 μl
Hot Start KlenTaq Buffer, 10x conc.1.2 ml5 x 1.2 ml

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.capfinal conc.1 assay @ 50 μl
PCR-grade Waterwhitefill up to 50 μl
10x reaction Buffergreen1x5 μl
dNTP Mix 10 mMwhite200 μM each1 μl
KlenTaqred1.25 - 2.5 units/reaction0.25 - 0.5 μl
forward primer
(10 μM)
0.1 - 0.5 μM0.5 - 2.5 μl
reverse primer
(10 μM)
0.1 - 0.5 μM0.5 - 2.5 μl
template DNA10 pg - 1 μg1)

1)genomic DNA: 1 ng - 1 μg, plasmid and viral DNA: 1 pg - 1 ng

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 °C10 - 12 min1x
denaturation
annealing2)
elongation3)
95 °C
50 - 68 °C
72 °C
15 - 30 sec
15 - 30 sec
30 sec - 4 min

25 - 35x

2)The annealing temperature depends on the melting temperature of the primers used.
3)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 optimisation of the recommended parameters may be necessary for each new template DNA and/or primer pair.

Related products: Mg2+ Stock, #PCR-266