<address id="rnhrh"><form id="rnhrh"></form></address>

      <form id="rnhrh"></form>

                    <form id="rnhrh"></form>

                    Products > Virus-related Products > HS Hotstart Taq DNA Polymerase P1091

                    HS Hotstart Taq DNA Polymerase P1091

                    HS Hotstart Taq DNA Polymerase

                    For research use only

                    Components  

                    Component

                    P1091 (500 U)

                    10 × HS Hotstart Taq Buffer (Mg2+ plus)

                    1 ml × 2 

                    dNTP Mix (10 mM each)

                    400 μl  

                    HS Hotstart Taq DNA Polymerase (5 U/μl)

                    100 μl

                     

                    Storage

                    This reagent should be kept at -30~15°C.

                     

                    Description

                    HS Hotstart Taq DNA Polymerase is a hot-start Taq DNA polymerase produced by mixing Taq antibody with Taq DNA polymerase in optimum proportion. Based on the thermostable properties of Taq antibody, the activity of HS Hotstart Taq DNA Polymerase remains strictly closed at 55°C, minimizing non-specific amplification during the mixing and heating phases of the reaction system. When the reaction is maintained above 30 sec at 95°C, Taq antibody is completely inactivated and the polymerase activity is fully released, ensuring the extremely high amplification sensitivity and specificity of the PCR system. The activation of HS Hotstart Taq DNA Polymerase is not affected by buffer pH, ion strength or other factors. It is suitable for various hot-start PCR and qPCR reactions based on Taq DNA polymerase. It can be used to amplify low-copy genes from complicated templates (genome, cDNA) and is the preferred hot-start Taq DNA polymerase for molecular diagnostic reagents based on PCR/qPCR. The PCR product has 3 '-d A ends and can be cloned to T vector.

                     

                    Unit Definition

                    Using activated salmon sperm DNA as template/primer, the activity of ingesting 10 nmol total nucleotides as acid insoluble substances within 30 min at 74°C is defined as 1 unit of activity (U).

                     

                    Quality Control

                    Detection of closure of activity: in 1 × Champagne Taq Buffer, the reaction was conducted at 65°C for 30 min, and the released activity was < 5%.

                    Detection of release of activity: in 1 × Champagne Taq Buffer, after heating at 95°C for 30 sec, and the released activity was > 95%.

                    Exonuclease residue detection: 50 U of this polymerase and 50 pmol ssDNA and dsDNA substrate were incubated at 37°C for 16 h, and the DNA bands did not change after denaturation PAGE.

                    Detection of endonuclease residue: 50 U of this polymerase and 0.3 μg of pBR322 DNA were incubated at 37°C for 4 h, and the DNA bands of the plasmids were not changed by agarose gel electrophoresis.

                    RNase residue detection: 50 U of this polymerase and 1 μg of 293 cell RNA were incubated at 37°C for 30 min, and the RNA bands was unchanged by agarose gel electrophoresis.

                    E.coli DNA residue detection: the nucleic acid residue in 50 U of this polymerase was detected by E. coli gDNA-specific TaqMan qPCR, and the residue of E. coli genome was less than 10 copies.

                    Functional detection: In the 20 μl PCR system, cDNA corresponding to the total RNA of 1 pg-200 ng HeLa cells was used as the template to amplify 4 different gene loci. The gradient of PCR product yield could be detected by fluorescence after 35 cycles. At a minimum, the corresponding amplification products were obtained from cDNA corresponding to the total RNA of 1 pg HeLa cells.

                     

                    Protocol

                    Reaction system

                    Component

                    Amount

                    10 × HS Hotstart Taq Buffer (Mg2+ plus)a

                    5 μl

                    dNTP Mix (10 mM each)

                    1 μl

                    Primer 1 (10 μM)

                    2 μl

                    Primer 2 (10 μM)

                    2 μl

                    Template DNAb

                    x μl

                    HS Hotstart Taq DNA Polymerase (5 U/μl)c

                    0.5 μl

                    ddH2O

                    to 50 μl

                    a. For most PCR reactions, the optimal final concentration of Mg2+ is 1.5-2 mM. Mg2+ with a final concentration of 2 mM has been included in the system. If necessary, the optimal concentration of Mg2+ can be explored with 25 mM MgCl2 at intervals of 0.2-0.5 mM.

                    b. The optimal reaction concentration of different templates varies, and the table below shows the recommended amount of 50 μl reaction system templates.

                    Human genome DNA

                    1-500 ng

                    E.coli genome DNA

                    1-100 ng

                    λ DNA

                    0.1-10 ng

                    Plasmid DNA

                    0.1-10 ng

                    c. The amount of enzyme can be adjusted between 0.125-0.5 μl. In general, increasing the amount of enzyme can increase the amplification yield, but it may decrease the specificity.

                    * When the GC content of the amplified fragment is > 60%, and normal amplification cannot be achieved after optimizing the conditions, PCR Enhancer (DSBio #P9041) is recommended to optimize PCR reaction.

                     

                    Reaction procedure

                    Temperature

                    Time

                    Cycle

                    95°C

                    30 sec (initial denaturation)

                    /

                    95°C

                    30 sec

                    30~35

                    55°C*

                    30 sec

                    72°C

                    60 sec/kb

                    72°C

                    7 min (final extension)

                    /

                    * The annealing temperature should be adjusted according to the Tm value of the primer, which is generally set to be 3-5°C lower than the Tm value of the primer.


                    正规手机购彩 <蜘蛛词>| <蜘蛛词>| <蜘蛛词>| <蜘蛛词>| <蜘蛛词>| <蜘蛛词>| <蜘蛛词>| <蜘蛛词>| <蜘蛛词>| <蜘蛛词>| <蜘蛛词>| <蜘蛛词>| <蜘蛛词>| <蜘蛛词>| <蜘蛛词>| <蜘蛛词>| <蜘蛛词>| <蜘蛛词>| <蜘蛛词>| <蜘蛛词>| <蜘蛛词>| <蜘蛛词>| <蜘蛛词>| <蜘蛛词>| <蜘蛛词>| <蜘蛛词>| <蜘蛛词>| <蜘蛛词>| <蜘蛛词>| <蜘蛛词>| <蜘蛛词>| <蜘蛛词>| <蜘蛛词>| <蜘蛛词>| <蜘蛛词>| <蜘蛛词>| <蜘蛛词>| <蜘蛛词>| <蜘蛛词>| <蜘蛛词>| <蜘蛛词>| <文本链> <文本链> <文本链> <文本链> <文本链> <文本链>