DNA Amplification and PCR

Taq DNA Polymerase, recombinant

31,00 379,00 

Taq DNA Polymerase is now supplied with -inhibitor free- DNA Loading buffer IF

  • Best choice for colony PCR
  • Adds a single deoxyadenosine (A) to the 3′- ends of PCR products
  • No detectable 3’→5′ proofreading exonuclease activity
  • For standard PCR where proofreading is not required
  • Excellent cost-performance ratio 

Concentration: 5U/µL
Source: Purified from an E. coli strain carrying a plasmid with Taq DNA polymerase gene from Thermus aquaticus YT-1.
Reagents supplied with: 10x Taq polymerase buffer and 10x DNA Loading buffer IF -inhibitor free-
Store at: -20 °C

Lieferzeit: Lieferzeit 1-2 Werktage

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Beschreibung

The Simplebiotech Taq Polymerase is purified from an E. coli strain carrying a plasmid with Taq DNA polymerase gene from Thermus aquaticus YT-1.

Description: Taq DNA Polymerase is a thermostable enzyme that catalyses 5’→3′ synthesis of DNA. The enzyme has no 3’→5’ proofreading exonuclease activity but possesses low 5’→3′ exonuclease activity.

Unit definition: One unit is defined as the amount of enzyme required to catalyse the incorporation of 10 nmoles of dNTPs into acid insoluble material in 30 minutes at 72 °C.

Assay conditions: The polymerase activity was assayed in 10 mM Tris-HCl (pH 8.5 at 25 °C), 50mM KCl, 1.5 mM MgCl2, 200 µM each of dATP, dGTP, dCTP, dTTP (a mix of unlabelled and [3H] dTTP) and 12.5 µg activated calf thymus DNA, in a final volume of 50 µL.

Storage buffer: 100 mM NaCl, 50 mM Tris-HCl (pH 8.0 at 25 °C), 1mM DTT, 0.1 mM EDTA, 1% Triton X-100 and 50% Glycerol.

Taq DNA polymerase buffer: 50 mM KCl, 10 mM Tris-HCl (pH 8.5 at 25 °C) 1.5 mM MgCl2, 0.1 % Triton X-100.

Quality control:

Our Taq DNA Polymerase has been extensively functionally tested and carefully tested for absence of endo- and exodeoxyribonucleases.

Recommended PCR set-up:

Thaw buffer, dNTPs and primers and put them back on ice. Keep all components including the reaction mix on ice during the hole PCR set-up. Always include a “no template control” including all components except template DNA and if possible, a positive control. The 10x Taq Polymerase buffer can be replaced for the “5x Magic orange” direct load buffer.

Component

Amount

Final concentration

10x Taq Polymerase buffer

5µL (or 10µL Magic orange)

1x

dNTP 10mM (each)

1µL

200µM (each)

Forward Primer 10µM

2.5µL

0.5µM

Reverse Primer 10µM

2.5µL

0.5µM

Template DNA

Variable (1ng plasmid or 100ng genomic)

 

Taq Polymerase

0.25µL

1.25U

Nuclease free water

up to 50µL

 

Total

50µL

 

 

Vortex gently and spin down.

Manual hot start can be simply achieved by keeping PCR mix on ice, until the thermal cycler has reached 94 °C. Then place the PCR tubes into the thermal cycler.

 

Step

Tepmperature

Time

Number of cycles

Initial denaturation

94 °C

3min

1x

Denaturation

94 °C

30s

 

25-35x

Annealing

45-72 °C1

30s

Elongation

72 °C

30s – 4min2

Final elongation

72 °C

7min

1x

                                         
1) The recommended annealing temperature is 5 °C below the calculated melting temperature of the primer with the lower melting temperature.

2) Use 1min per kb of PCR product.

Calculating melting Temperature of Primers:

For primers shorter than 20 nucleotides melting temperature (Tm) can be approximated by the following equation:

Tm= 4 (G + C) + 2 (A + T),

G, C, A, T represent the number of the respective nucleotides in the primer sequence.

For longer primers thermodynamic calculations taking into account base stacking energy for calculation are recommended. This can be done easily using free online oligo calculation programs (e. g. https://www.biophp.org/minitools/melting_temperature/demo.php?formula=basic

http://biotools.nubic.northwestern.edu/OligoCalc.html).

Cloning of PCR Products: Taq Polymerase generates 3’ single-base dA overhangs that can be used for direct cloning into TA cloning vectors. For direct cloning into blunt vectors blunting of the purified PCR fragment (e. g. using the Klenow enzyme) is strongly recommended.

Licenses/Patents/Disclaimers:
The PCR process is disclosed and claimed in European patents EP0201184B1 and EP0200362B1 or United States patent US4965188A, which have expired along with all family members/equivalents in all territories. However, some specific product applications may fall within the scope of protection of granted patents, which may be in force in the United States, European Union and certain countries. The purchase of this product does not include a license to perform any application subject to patent protection, it is the sole responsibility of the buyer to ensure that use of the product does not infringe the patent rights of third parties.
For Research Use Only. Not for use in diagnostic procedures.

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