Taq DNA Polymerase
 
Product Taq DNA Polymerase (New Buffer!) Item Number: TAQ500 Conc: 5 units/1"l

Description:

Taq DNA Polymerase is a thermostable DNA Polymerase isolated from a strain of Thermus aquaticus. Taq has a half life of 3 hours at 95 °C, it is very stable. Taq has high fidelity with an error frequency 10 / 106 (or 0.01 / 103) during DNA synthesis. Taq is designed for use in primer extension reaction. DNA sequencing at high temperature may decrease the second structure of some DNA templates and permit polymerization through base-paired region. DNA sequencing with Taq DNA Polyinerase produces uniform bands intensities and low background

Performance & Quality Testing:

Taq DNA Polymerase is highly purified free of contaminating endonucleases, exonucleases and nicking activity. For endonuclease assay, 1 "g of Lambda I Hind III DNA is incubated with 20 units of the enzyme in assay buffer at 75º C for 16 hrs and no visible contaminating activity is observed; For exonucleases assay, 1 "g of pBR322 plasmid DNA is incubated with 10 units of enzyme for 16 hrs at 75º C in assay buffer and no detectable exonuclease is observed. The purity of the enzyme is also evaluated by adding 10 units of Taq DNA Polymerase in 100 "l of a reaction mixture for making first strand cDNA at beginning and no impaired effect on the first strand is observed.

Unit Definition:

One unit incorporates 10 nmole of dNTP into acid-insoluble material in 30 mm. at 74º C. Concentration in Storage Buffer: 5 units / p1 in 100mM KC1, 20 mM Tris HC1 (pH 8.0, 22°C ), 0.lmM EDTA. 0.5mM PMSF, 1mM DTT, 50% glycerol.

10 X Taq Reaction Buffer: (New!)

100 mM KC1, 100mM (NH4)2SO4, 200 mM Tris HC1 (pH 8.75) at 22° C, 1% Triton X-100 and 1mg/ml BSA. Buffer is optimized for use with 200 "M dNTPs.

Magnesium Sulfate: (New!)

20mM MgSO4. The final MgSO4 may be variable according to requirements. Normally 2mM MgSO4 is recomemded.

General Reaction mixture for PCR:


Taq (5u/ul): 0.5ul, l0xRxn Buffer: l0ul, MgSO4 (20mM): l0ul, dNTP mixture (2.5mM each): 8u1, Primer 1: 0.2-1.0um, Primer 2 : 0.2-1 .0uM, Template : 10pg-lug, Sterilized ddH2O up to l00ul

Primer Extension Characteristics:


Taq has the independent terminal transferase activity which results in the addition of a single nucleotide ( adenosine ) at 3’ end of the extension product. TA cloning vector is recommended if the extension product is needed to be cloned.

Reaction Mixture Set Up
Reagent Final Concentration Quantity Reagent Positive Control Negative Control
Water (PCR--Grade) --- Variable Water (PCR--Grade) 32.8"l 33. 8"l
10x Taq reaction buffer 1x 5"l 10x Taq reaction buffer 5"l 5"l
MgSO4(20mM) 2-4mM variable MgSO4(20mM) 5"l 5"1
2.5mM dNTP mixture 200"M of each 4"1 2.5mM dNTP mixture 4"l 4"l
Primer I, forward 0.l-1"M variable Primer I(l0"M), forward l"l 1"l
Primer II, reverse 0.1-1 "M variable Primer II(l0"M), reverse 1 "1 1 "1
Taq DNA polymerase 1-1.5u/50"l variable Taq DNA polymerase(5u/"l) 0.2"1 0.2"l
Template DNA See note 1 variable Control DNA Template 1 "1 ---
Total Volume --- 50"l Total Volume 50"l 50"l
  • Gently vortex the sample and briefly centrifuge to collect all drops from walls of the tube.
  • Overlay the sample with one-half of the total reaction volume of mineral oil or add an appropriate amount of wax. This step may be omitted if the thenno cycler is equipped with a heated lid.
  • Place samples in a thermo cycler and start PCR.

Note for the Components of the Reaction Mixture:
  • Template DNA: Usually the amount of template DNA is in the range of 0.01-lug plasmid or phage DNA and 0.1- 1"g for genomic DNA, for a total reaction mixture of 50"l.
  • Primers: The PCR primers are usually 15-30 nucleotides in length, longer primers provide higher specificity. The GC content of primer should be 40-60%. The primer should not be self-complementary or complementary to any other primer in the reaction mixture, and the melting temperature of flanking primers should not differ by more than 5°C. If the primer is shorter than 25 nucleotides, the approx. melting temperatureTM is calculated using the formula as: Tm=4(G+C) + 2(A+T).
  • MgSO4 concentration: Since Mg2+ ions form complex with dNTPs, primers and DNA templates, the optimal concentration of MgSO4 has to be selected for each experiment. In our experiments, at a final dNTP concentration of 200"M. 2mM MgSO4 concentration is suitable in most case.
  • dNTPs: The final concentration of each dNTP in the reaction mixture is usually 200"M.
  • Taq DNA polymerase: Usually 1-1.5u of Taq DNA polymerase are used in the 50"l of reaction mix. Higher Taq DNA polymerase concentrations may cause synthesis of nonspecific products. However, if inhibitors are present in the reaction mix (e.g., if the template DNA used is not highly purified), higher amounts of Taq DNA polymerase(2-3u)may be necessary to obtain a better yield of amplification products.
  • Cycling conditions: Usually denaturation for 0.5-2min at 94-95°C is sufficient; the optimal annealing temperature is 5°C lower than the melting temperature of primer-template DNA duplex; Usually the extending step is performed at 70-75°C. Recommended extending time is 1min for the synthesis of PCR fragments up to 2kb. When larger DNA fragments are amplified, the extending time is usually increased by 1min for each 1kb.
  • Number of cycles: The number of PCR cycles depends on the amount of template DNA in the reaction mix and on the expected yield of the PCR product. For less than 10 copies of template DNA, 40 cycles should be performed. if the initial quantity of template DNA is higher, 25- 35 cycles are usually sufficient.
  • Final extending step: After the last cycle, the samples are usually incubated at 72°C for 5-15min to fill-in the protruding ends of newly synthesized PCR products. Also, during this step, the terminal transferase activity of Taq DNA polymerase adds extra A nucleotides to the 3’-ends of PCR products.