Taq Polymerase / Labeling Buffer

For general laboratory use.

Definição de unidade: 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.

Envio: shipped on gel packs

Condições de armazenamento: store at -20 °C
avoid freeze/thaw cycles

Validade: 12 months

Forma: liquid

Concentração: 5 units/μl

Descrição:
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.

Contente:
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
component||PCR-201S||PCR-201L
Taq Polymerase||200 units / 40 μl||5x 200 units / 5x 40 μl
Labeling Buffer||1.2 ml||5 x 1.2 ml
MgCl2 Stock||1.5 ml||2 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.||cap||stock conc.||final conc.||1 assay @20 μl||1 assay @ 50 μl
PCR-grade Water||white||||||fill up to 10 μl||fill up to 30 μl
Labeling Buffer||green||10x||1x||2 μl||5 μl
dNTP Mix / 10 mM #NU-1006||white||10 mM||200 μM||0.4 μl||1 μl
Taq Polymerase||red||5 units/μl||0.025 units/μl||0.1 μl||0.25 μl
primer mix or each primer||||10 μM each primer||200 - 400 nM each primer||0.4-0.8 μl||1 - 2 μl
template/sample DNA||||||||10 μl < 10 ng DNA||20 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.
initialdenaturation||95 °C||2 min||1x
denaturationannealing1)elongation2)||95 °C50 - 68 °C72 °C||10 - 20 sec10 - 20 sec20 sec - 4 min||25 - 35x1)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-274 - #PCR-276) 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 volume||50 μl final assay volume
2 mM||-||-
3 mM||0.8 μl||2.0 μl
4 mM||1.6 μl||4.0 μl
5 mM||2.4 μl||6.0 μl

Produtos relacionados:

• dNTP Mix / 10 mM, #NU-1006
• Ruby Buffer, #PCR-272
• Crystal Buffer, #PCR-271
• KCl Buffer, #PCR-262
• SYBR DNA Stain, #PCR-273
• Gel Loading Buffer with DNA Stain: Blue #PCR-273, Green #PCR-275, Orange #PCR-276
• Mg2+ Stock, #PCR-266