Premixed Ready-to-Use Solution Long Taq Mix PCR Master Mix

Description
Long Taq 2X Mix is a premixed, ready-to-use solution containing Long Taq DNA Polymerase, dNTPs, MgCl₂ and Reaction Buffer at optimal concentrations for efficient amplification of DNA templates by PCR. To prepare the final PCR, only primers and template DNA are added. Long Taq Mix contributes to highly reproducible PCR by reducing the risk of pipetting errors, miscalculation and contamination. It also contributes to higher sensitivity by adding enhancer.
 

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

Features
Convenient : only primers and template DNA are added when prepare final PCR
High efficiency: saving your time by simplifying the process 
Reproducible: reducing the risk of pipetting errors, miscalculation and contamination

Applications
PCR for long templates of up to 40kb
High reproducible ,high throughput PCR for complex templates

Unit Definition
One unit is defined as the amount of the enzyme required to catalyze the incorporation of 10 nM of dNTPs into an acid-insoluble form in 30 minutes at 70°C using hering sperm DNA as substrate.

2×PCR Long Taq Mix
20 mM Tris-HCl (pH 8.0), 100 mM KCl , 3 mM MgCl₂, 400 μM dNTPs, 0.1 U/μl Long Taq DNA Polymerase, bromophenol blue

Basic PCR Protocol
1. Add the following components to a sterile 50 μl microcentrifuge tube sitting on ice:

2. Mix contents of tube and overlay with 50 μl of mineral or silicone oil.
3. Cap tubes and centrifuge briefly to collect the contents to the bottom.
4. Incubate tubes in a thermal cycler at 94°C for 3 minutes to completely denature the template.
5. Perform 25-35 cycles of PCR amplification as follows:

6. 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.

Notes on cycling conditions

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

  Denaturation for 30 sec to 2 min at 94-95ºCis sufficient. If the amplified DNA has a very high GC content, denaturation time may be increased up to 3-4 min.

  Optimal annealing temperature is 5ºC 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 1-2ºC.

  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. 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 extension step can be extended for amplicons that will be cloned into T/A vectors.

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

Store all components at -20°C