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Taq Polymerase / Labeling Buffer

Thermostable DNA Polymerase

Thermus aquaticus, recombinant, E. coli

Cat. No. Amount Price (EUR) Buy / Note
PCR-201S 200 units 44,00 Add to Basket/Quote Add to Notepad
PCR-201L 1000 units 176,00 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 using hering sperm DNA as substrate.

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:
Taq Polymerase / Labeling Buffer is recommended for DNA labeling or mutagenesis. The buffer system is specially optimized for incorporation of labeled or modified nucleotides into DNA. It gives superior results in a broad range of reaction conditions with most primer-template pairs but amplification may also tend to an increased unspecifity.
The enzyme replicates DNA at 72 °C. It catalyzes the polymerization of nucleotides into duplex DNA in 5'→3' direction in the presence of magnesium. It also possesses a 5'→3' polymerization-dependent exonuclease replacement activity but lacks a 3'→5' exonuclease (proof-reading) activity.

Content:
Taq Polymerase (red cap)
5 units/μl Taq DNA Polymerase in 20 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 8.0 (25°C)

Labeling Buffer (green cap)
10x conc. complete PCR buffer containing 600 mM Tris-HCl, 150 mM (NH4)2SO4, 20 mM MgCl2, 0.05 % Tween-20, 0.05 % Nonidet P-40, pH 8.8 (25°C)

MgCl2 Stock Solution (yellow cap)
25 mM MgCl2

componentPCR-201SPCR-201L
Taq Polymerase200 units / 40 μl1000 units / 200 μl
Labeling Buffer1.2 ml5 x 1.2 ml
MgCl2 Stock1.5 ml4 x 1.5 ml


Assay Set-Up:
Before starting, vortex all components thoroughly to ensure homogeneity.
Prepare a premix for the number of assays you need according to the following protocol:

comp.capstock conc.final conc.1 assay @20 μl1 assay @ 50 μl
PCR-grade Waterwhitefill up to 10 μlfill up to 30 μl
Labeling Buffergreen10x1x2 μl5 μl
dNTP Mix / 10 mM #NU-1006white10 mM200 μM0.4 μl1 μl
dNTP Mix / 10 mM #NU-1006white10 mM200 μM0.4 μl1 μl
Taq Polymerasered5 units/μl0.025 units/μl0.1 μl0.25 μl
primer mix or each primer10 μM each primer200 - 400 nM each primer0.4-0.8 μl1 - 2 μl
template
/sample DNA
10 μl < 10 ng DNA20 μl < 20 ng DNA

Select PCR tubes, stipes or plates as recommended for your cycler model.
Aliquot premix into each well and add template DNA (or PCR-grade Water for negative controls).

Cycling Conditions:
Spin down the tubes/plate briefly to remove bubbles and place them into the cycler.

initial
denaturation
95 °C2 min1x
denaturation
annealing1)
elongation2)
95 °C
50 - 68 °C
72 °C
10 - 20 sec
10 - 20 sec
20 sec - 4 min

25 - 35x
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.

Additional Buffer Systems:
Ruby Buffer (#PCR-272) includes a density reagent + tracking dye and allows the direct loading of the PCR products into a electrophoresis gel. For DNA detection / fluorescent DNA staining we recommend to use new generations dyes (i.g. SYBR DNA Stain, #PCR-273) instead of the classical but highly mutagenic ethidium bromide.
Crystal Buffer(#PCR-271) is recommended for down-stream applications such as DNA sequencing, ligation, restriction digestion or where an analysis of the PCR product by absorbance or fluorescence excitation is required. For gel electrophoresis add gel loading buffer and fluorescent DNA stain (i.g. Gel Loading Buffer with DNA Stain, #PCR-255) before loading the PCR into the gel. Using pre-stained gels or post-run staining protocols is also possible.
KCl Buffer (#PCR-262) is recommended for use in routine PCR reactions. The buffer is optimized for highest specificity but may require additional fine-tuning of assay parameters like MgCl2 concentration and annealing temperature.

Optimization of MgCl2 concentration:
A final Mg2+ concentration of 2.0 mM is recommended in combination with Labeling Buffer. However, if an individual Mg2+ optimization is essential add 25 mM MgCl2 stock solution (#PCR-266) as shown in the table below.

final MgCl2 conc.20 μl final assay volume50 μl final assay volume
2 mM--
3 mM0.8 μl2.0 μl
4 mM1.6 μl4.0 μl
5 mM2.4 μl6.0 μl

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