* with dNTPs mixture
Long Taq DNA Polymerase has two Concentrations with 2.5 U/μl and 5 U/μl, default package is 5 U/μl.
10×Long PCR Buffer (Mg2+ Plus) can replace with 10×PCR Buffer (Mg2+ free) and 25 mM MgCl2. Please choose the appropriate package for your experiment.
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
? PCR amplification of DNA fragments as long as 20 kb
? DNA labeling
? DNA sequencing
? PCR for cloning
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.
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 Taq Buffer Ⅰ with Mg2+
500mM Tris-HCl pH 8.8，160mM (NH4)2SO4 ，25mM MgCl2 ，1% Triton X-100
10X Long Taq Buffer Ⅱ with Mg2+
200mM Tris-HCl PH8.8，100mM KCl，100mM (NH4)2SO4，16mM MgSO4，1% Tritonx-100
? High fidelity: three times fidelity of Taq DNA Polymerase.
? Longer fragment: amplify long templates as long as 20kb.
? Amplification of complex template (GC rich or repetitive sequence).
? Generates 3'-dA and blunt end PCR products.
? 10xLong PCR BufferⅠis classical Long Taq DNA Polymerase buffer, is good for long template especially above 10kb.
? 10xLong PCR Buffer Ⅱ is a special buffer optimized by Dongsheng Biotech. It is for better fidelity but not good at long template above 10kb.
? Users could choose suitable buffer 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:
1.1 Recommended PCR assay with PCR Buffer (Mg2+ plus)
4-16 μl PCR Enhancer can be added to the reaction system of 50 μl. By reducing the dissociation temperature of DNA template and promoting the effective amplification of DNA template, PCR Enhancer can increase the sensitivity and specificity of PCR reaction.
1.2 Recommended PCR assay with PCR Buffer (Mg2+ free )
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:
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. An alyze the amplification products by agarose gel electrophoresis and visualize by ethidium bromide staining. Use appropriate molecular weight&nb, sp;standards.
Notes on cycling conditions
l Initial denaturation can be performed over an interval of 1~5 min at 95℃ depending on the GC content of template.
l 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.
l 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℃.
l The number of PCR cycles depends on the amount of emplate DNA in the reaction mix and on the expected yield of the PCR product.&, nbsp;2535 cycles&nbs, p;are usually sufficient
for the majority PCR reaction. Low amounts of starting template may require 40 cycles.
l The time of the final extensi, on step can be extended for amplicons that will be cloned into T/A vectors.
Guidelines for preventing contamination of PCR reaction
During PCR more than 10 million copies of template DNA are generated. Therefore, care must be taken to avoid contamination with other templates and amplicons that may be
present in the laboratory environment. General recommendations to lower the risk of contamination are as follows:
? Prepare your DNA sample, set up the PCR mixture,perform thermal cycling and analyze PCR products in separate areas.
? Set up PCR mixtures in a laminar flow cabinet equipped with an UV lamp.
? Wear fresh gloves for DNA purification and reaction set up.
? Use reagent containers dedicated for PCR. Use positive displacement pipettes, or use pipette tips with aerosol filters to prepare DNA samples and perform PCR set up.
? Always perform “no template control” (NTC) reactions to check for contamination