ELISA Long PCR Taq DNA polymerase

Long PCR Taq DNA Polymerase,a combination of two thermostable DNA polymerases, Taq and Pfu, is a special formµLation designed for amplifying large fragment. This specially formµLated 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 resµLts 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 Ⅰ with Mg2+, 10X Long PCR Taq Buffer Ⅱ with Mg2+ Applications • PCR amplification of DNA fragments any Quantitys 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 DTT，0.1% NP-40, 0.1% Tween20, 0.2mg/mL BSA, 50% (v/v) glycerol 10X Long PCR Taq Buffer Ⅰ with Mg2+ 500mM Tris-HCl pH 8.8，160mM (NH4)2SO4 ，25mM MgCl2 ，1% Triton X-100 10X Long PCR Taq Buffer Ⅱ with Mg2+ 200mM Tris-HCl PH8.8，100mM KCl，100mM (NH4)2SO4，16mM MgSO4，1% 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 BufferⅠis classical Long PCR Taq DNA Polymerase buffer, is good for long template especially above 10kb. 10xLong PCR Buffer Ⅱ 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 microcentrifµge 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 centrifµge 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 molecµLar 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.