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                    Products > PCR Products > OptimusTM Hotstart Taq Mix P2042 1ML X 5

                    OptimusTM Hotstart Taq Mix P2042 1ML X 5

                    2× OptimusTM Hotstart Taq Mix is a premixed, ready-to-use solution containing Hotstart Taq DNA Polymerase, dNTPs, Mg2+ and Reaction Buffer at optimal concentrations for efficient amplification of DNA templates by PCR. To prepare the final PCR, only need to add primers and template DNA.

                    OptimusTM Hotstart Taq Mix

                    #P2042, 5 ml

                      

                    Contents:

                    2× OptimusTM Hotstart Taq Mix

                    1 ml × 5

                    Nuclease-free water

                    1 ml × 5 

                     

                    Store at -20°C

                    For research use only.

                    In total 10 vials.

                     

                    Description

                    2× OptimusTM Hotstart Taq Mix is a premixed, ready-to-use solution containing Hotstart Taq DNA Polymerase, dNTPs, Mg2+ and Reaction Buffer at optimal concentrations for efficient amplification of DNA templates by PCR. To prepare the final PCR, only need to add primers and template DNA. This premixed formulation saves time and reduces contamination due to the fewer pipetting steps required for PCR set-up. The mix retains all features of Hotstart Taq DNA Polymerase.

                    OptimusTM Hotstart Taq DNA Polymerase is a hot-start polymerase with chemical modification, which brings higher specificity by reducing non-specific products as the enzyme activity is temperature-dependent and is inhibited at room temperature. The amplification length and speed can reach to 5 kb (simple template) and 2min/kb (simple template up to 20s/kb) separately. Hotstart Taq has 5-3 polymerase activity, but no 3-5 exonuclease activity. The product of Hotstart Taq has overhanged dA at 3-end. OptimusTM Hotstart Taq DNA Polymerase has zero animal source pollution by being produced with advanced chemical modification. And it is much more stable than antibody-modified hot-start polymerase. Its efficiency is higher than most chemical-modified polymerases and the initial-denaturation time can be reduced to 3 minutes.

                     

                    PRODUCT USE LIMITATION.

                    This product is developed, designed and sold exclusively for research purposes and in vitro use only. The product was not tested for use in diagnostics or for drug development, nor is it suitable for administration to humans or animals.

                    Unit Definition

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

                    Composition of the 2× OptimusTM Hotstart Taq Mix

                    0.5U/ul OptimusTM Hotstart Taq DNA Polymerase, 2× PCR buffer, 0.4mM dNTPs, 3.2mM MgCl2, 0.02% bromophenol blue.

                    Applications

                    ? High-specificity amplification & multiplex PCR: hot-start polymerase with chemical modification

                    ? Thermostable: half-life over 40 min at 95°C incubation

                    ? Generates 3'-dA overhangs PCR products

                     

                    Basic PCR Protocol

                     

                    1. Add the following components to a sterile microcentrifuge tube sitting on ice (not necessary):

                    Reagent

                    Quantity

                    Final concentration

                     OptimusTM Hotstart Taq Mix

                    25 μl

                    Forward Primer

                    variable

                    0.4-1 μM

                    Reverse Primer

                    variable

                    0.4-1 μM

                    Template DNA

                    variable

                    10pg-1μg

                    Water, nuclease-free

                    to 50 μl

                     

                     

                    Recommendation amounts of template DNA in a 50 μl reaction mix:

                    Human genomic DNA

                    0.1μg-1μg

                    Plasmid DNA

                    0.5ng-5ng

                    Phage DNA

                    0.1ng-10ng

                    E.coli genomic DNA

                    10ng-100ng

                     

                    2. Mix contents in the 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 minutes

                    25-35 Cycles

                    94°C

                    55-68°C

                    72°C

                    30 seconds

                    30 seconds

                    1-2 minutes

                    Final Extension

                    72°C

                    10 minutes

                     

                    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 nucleic acid dye staining. Use appropriate molecular weight standards.

                    Notes on cycling conditions

                    OptimusTM Hotstart Taq DNA Polymerase adopts improved chemical modification technology, so it relies on temperature to activate the polymerase activity, which can effectively inhibit non-specific bindings, and the reaction system can be configured at room temperature.

                    The half-life of OptimusTM Hotstart Taq DNA Polymerase is >40 minutes at 95°C.

                    OptimusTM Hotstart Taq DNA Polymerase accepts modified nucleotides (e.g. biotin-, digoxigenin-, fluorescent-labeled nucleotides) as substrates for the DNA synthesis.

                    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 reactions. Low amounts of starting template may require 40 cycles.

                    Guidelines for preventing contamination of PCR

                    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.

                    Quality Control

                    The absence of endodeoxyribonucleases, exodeoxyribonucleases and ribonucleases is confirmed by appropriate quality tests. Functionally tested in amplification of a single-copy gene from human genomic DNA.

                    Endodeoxyribonuclease Assay

                    No detectable conversion of covalently closed circular DNA to a nicked DNA was observed after incubation of 25 μl OptimusTM Hotstart Taq Mix (2×) with 1μg pBR322 DNA for 4 hours at 37°C and 70°C.

                    Exodeoxyribonuclease Assay

                    No detectable degradation of lambda DNA-HindIII fragments was observed after incubation of 25 μl OptimusTM Hotstart Taq Mix (2×) with 1μg digested DNA for 4 hours at 37°C and 70°C.

                    Ribonuclease Assay

                    0% of the total radioactivity was released into trichloroacetic acid-soluble fraction after incubation of 25 μl OptimusTM Hotstart Taq Mix (2×) with 1μg E.coli [3H]-RNA (40000cpm/μg) for 4 hours at 37°C and 70°C.


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