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Taq DNA Polymerase
Code:P1011/P1012/P1013/P1014/P1015
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Taq DNA Polymerase

Catalog #

AmountU

Price

P1011

500

 

P1012*

500

 

P1013

1,000

 

P1014*

1,000

 

P1015

18,000

 

 


Contents

P1011

Taq DNA Polymerase (5 U/μl)

100 μl

10×PCR Buffer (Mg2+ Plus)

1.25 ml

6×Loading Buffer

1 ml

 

P1012*

Taq DNA Polymerase (5 U/μl)

100 μl

10×PCR Buffer (Mg2+ Plus)

1.25 ml

dNTPs (each 2.5 mM)

1 ml

6×Loading Buffer

1 ml

 

P1013

Taq DNA Polymerase (5 U/μl)

200 μl

10×PCR Buffer (Mg2+ Plus)

1.25 ml×2

6×Loading Buffer

1 ml

 

P1014*

Taq DNA Polymerase (5 U/μl)

200 μl

10×PCR Buffer (Mg2+ Plus)

1.25 ml×2

dNTPs (each 2.5 mM)

1 ml×2

6×Loading Buffer

1 ml

 

P1015

Taq DNA Polymerase (5 U/μl)

100 μl×36

10×PCR Buffer (Mg2+ Plus)

1.25 ml×36


Note
Taq DNA Polymerase has two concentrations with 2.5 U/μl and 5 U/μl, default package is 5 U/μl.

10×PCR Buffer (Mg2+ Plus) can replace with 10×PCR Buffer (Mg2+ free) and 25 mM MgCl2.

Please choose the appropriate package for your experiment.

Description
Taq DNA Polymerase is purified from E. coli. expressing a cloned Thermus aquaticus DNA polymerase gene. This enzyme has a 5' → 3' DNA polymerase and a 5' → 3' exonuclease activity but lacks a 3' → 5' exonuclease activity. The enzyme consists of a single polypeptide with a molecular weight of approximately 94 kDa. Taq DNA polymerase is heat-stable and will synthesize DNA at elevated temperatures from single-stranded templates in the presence of a primer.

Applications
Standard PCR
DNA labeling
DNA sequencing
Numerous applications for which a high-quality, thermostable DNA polymerase is required

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.

Storage Buffer

20mM TrisCl ( pH8.0), 100mM KCl, 3.2mM MgCl1mM DTT0.1% Triton X-100 ,0.1% Tween20, 0.2mg/ml BSA, 50% (v/v) glycerol


10X PCR Buffer with Mg2+

 

100mM Tris-HCL PH8.8500mM KCl 16mM MgCl2 1% Triton100 



Note

ü    Recombinant Taq DNA Polymerase is the enzyme of choice for most PCR applications.

ü   The half-life of enzyme is >40 minutes at 95°C.

ü   The error rate of Taq DNA Polymerase in PCR is 2.2x10-5 errors per nt per cycle;

  

     the accuracy (an inverse of the error rate) an average number of correct nucleotides incorporated before making an error, is 4.5x104.

     

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

 

     synthesis.Compatible with TA cloning – generates PCR products with 3’-dA overhangs.

       

ü  Recommendations with Template DNA in a 50μl reaction volume

Human genomic DNA

0.1 μg-1 μg

Plasmid DNA

0.5 ng-5 ng

Phage DNA

0.1 ng-10 ng

E.coli genomic DNA

10 ng-100 ng


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

Components

Volume

Final Concentration

10×PCR Buffer (Mg2+ Plus)

dNTPs (each 2.5 mM)

Primer mix (each 10 μM)

Template DNA

Taq DNA Polymerase (5 U/μl)

5 μl

4 μl

2 μl

1–10 μl

0.1–0.5 μl

1 ×

0.2 mM each

0.4 μM each

n/a

0.5-2.5 U

Autoclaved distilled water

to 50 μl

n/a


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:

Step

Temperature

Duration

Initial Denaturation

94

3 minutes

25-35 Cycles

94℃

55-68℃

72℃

30 seconds

30 seconds

1 minutes

Final Extension

72

10 minutes


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

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

ü        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 1-2.

ü          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.

ü        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


 
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