Long PCR Taq DNA polymerase

Long PCR Taq DNA Polymerase, a combination of two thermostable DNA polymerases, Taq and Pfu, is a special formulation designed for amplifying large fragment. The Long PCR Taq was shown to amplify long templates from λ phage genome of up to 20 kb. It is also a better choice for amplifying complex template, such as GC-rich template.

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CAD$80.00

  • 250 U
  • 1000 U

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Long PCR Taq DNA Polymerase,a combination of two thermostable DNA polymerases, Taq and Pfu, is a special formulation designed for amplifying large fragment. This specially formulated Long PCR Taq was shown to amplify long templates from λ phage genome of up to 20 kb. It is also a better choice for amplifying complex template, such as GC-rich template.

Long PCR Taq is suitable as a direct replacement for ordinary Taq Polymerase in most applications. Using Long PCR Taq in your PCR reactions results in 3´-dA overhangs PCR products, which can be used in TA clone

Contents: Long PCR Taq DNA Polymerase, PCR Enhancer, 6x gel loading buffer, 10X Long PCR Taq Buffer with Mg2+, 10X Long PCR Taq Buffer with Mg2+

Applications

• PCR amplification of DNA fragments any sizes around 5 kb

• DNA labeling

• DNA sequencing

• PCR for cloning

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.

Storage Buffer

20mM TrisCl ( pH8.0), 100mM KCl, 3mM MgCl2 1mM DTT0.1% NP-40, 0.1% Tween20, 0.2mg/ml BSA, 50% (v/v) glycerol

10X Long PCR Taq Buffer with Mg2+

500mM Tris-HCl pH 8.8160mM (NH4)2SO4 25mM MgCl2 1% Triton X-100

10X Long PCR Taq Buffer with Mg2+

200mM Tris-HCl PH8.8100mM KCl100mM (NH4)2SO416mM MgSO41% Tritonx-100

Features

• High fidelity: three times fidelity of Taq DNA Polymerase.

• Longer fragment: amplify long templates as long as 40kb.

• Amplification of complex template (GC rich or repetitive sequence).

• Generates 3'-dA and blunt end PCR products.

Note:

  • 10xLong PCR Bufferis classical Long PCR Taq DNA Polymerase buffer, is good for long template especially above 10kb.
  • 10xLong PCR Buffer is an alternatie long PCR buffer . It is for better fidelity but may not be robust for longer templates above 10kb.

• Users may choose compare the two buffers for different template.

Basic PCR Protocol

The following basic protocol serves as a general guideline and a starting point for any PCR amplification. Optimal reaction conditions

(incubation time and temperature, concentration of Taq DNA Polymerase, primers, Mg2+, and template DNA) vary and need to be optimized.

1. Add the following components to a sterile microcentrifuge tube sitting on ice:

Reagent

Volume (50 µl rxn)

Final concentration

10x PCR Buffer

5 µl

1x

dNTPs(10 mM each)

1 µl

0.2 mM each

Primer I

Variable

0.4-1 µM

Primer II

Variable

0.4-1 µM

Long PCR DNA polymerase (5U/µl)

0.25-0.5 µl

1.25-2.5U/50 µl

Water

Variable to 50 µl

N.A.

2. Mix contents of tube. Cap tubes and centrifuge briefly to collect the contents to the bottom. When using a thermal cycler that does not

contain a heated lid, overlay the reaction mixture with 25 μl mineral oil.

3. Perform 25-35 cycles of PCR amplification as follows:

Initial Denaturation

94°C

3 min

25-35 Cycles

94°C

55-68°C

72°C

30s

30s

1-10 mins

Final Extension

72°C

10 min

4. Incubate for an additional 10 min at 72°C and maintain the reaction at 4°C. The samples can be stored at -20°C until use.

5. Analyze the amplification products by agarose gel electrophoresis and visualize by ethidium bromide staining. Use appropriate molecular weigh standards.

Notes on cycling conditions

- Initial denaturation can be performed over an interval of 1~5 min at 95 depending on the GC content of template.

-Denaturation for 30 sec to 2 min at 94~95 is sufficient. If the amplified DNA has a very high GC content, denaturation time may be increased up to 4 min.

-Optimal annealing temperature is 5 lower than the melting temperature of primer-temperature DNA duplex. If nonspecific PCR products are obtained optimization of annealing temperature can be performed by increasing temperature stepwise by 2.

-The number of PCR cycles depends on the amount of emplate DNA in the reaction mix and on the expected yield of the PCR products, 25-35 cycles are usually sufficient

for the majority PCR reaction. Low amounts of starting template may require 40 cycles.

-The time of the final extensi, on step can be extended for amplicons that will be cloned into T/A vectors.

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