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nTaq-Pure HOT (purified from insect cell)

No Product Name Cat.# Conc Size Price(₩) Add
1 nTaq-Pure HOT (purified from insect cell) P725PI 5 units/μl 250 units ₩ 144,000 추가
2 nTaq-Pure HOT (purified from insect cell) P750PI 5 units/μl 500 units ₩ 260,000 추가
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Product description

nTaq-Pure HOT (purified from insect cell) is a highly purified polymerase especially for PCR reactions where freedom from bacterial genomic DNA is essential. A gene encoding the Thermus aquaticus (Taq) DNA polymerase was cloned and expressed in Spodoptera frugiperda (Armyworm), and the enzyme was purified to homogeneity. nTaq-Pure HOT (purified from insect cell) is a hot start PCR polymerase which remains inactive at temperatures lower than 75. Nonspecific elongation of incorrectly annealed primers before initiation of PCR reaction is one major cause of nonspecific DNA amplification. For this reason, amplification of nonspecific bands can be effectively prevented by using nTaq-Pure HOT (purified from insect cell), thus increasing the specificity of target DNA amplification. In addition, nTaq-Pure HOT (purified from insect cell) was purified by state of the art technology for minimizing the E. coli genomic DNA contamination. PCR product was not detected after 40 cycles of amplification using E. coli 16S RNA primer.

 

Characteristics

- Absolutely free from E. coli genomic DNA contamination.

- Molecular weight: 94 kDa

- Error rate: 2.4 X 10-5

- Thermal stability: half life of 40 min at 95

- A-tail formation at 3’ ends of amplified duplex DNA.

- No activity at temperatures lower than 75. Heating up to 95 results in activation of enzyme.

 

Applications

- High specific amplification of DNA fragments shorter than 3kb.

- Amplification of cDNA and genomic DNA.

- Amplification of template DNA with secondary or higherordered structure that is resistant to PCR amplification

- Well-suited for an automated PCR machines, for which PCR reaction mixtures are prepared at room

  temperature

- Primer extension

- Multiplex PCR

 

Supplied with

- 10X nTaq-HOT buffer 

- dNTP Mixture (2 mM each)

- Sterile water

 

Quality control

- Purity: >99% on SDS-PAGE

- Endonuclease-free

- Exonuclease-free

- RNase free

- Inhibitor-free

 

Unit definition

One unit is defined as the amount of enzyme required to incorporate 10 nmol of dNTP into acid insoluble materials in 30 min at 74 in a 50-μl reaction mixture (20 mM Tris-HCl/pH 8.8, 50 mM KCl, 2.5 mM MgCl2, 10 mM β-mercaptoethanol, 12.5 μg of calf thymus DNA).

 

Storage buffer

20 mM Tris-HCl (pH 7.9), 100 mM KCl, 0.1 mM EDTA, 1mM DTT, 0.5% NP-40, 0.5% Tween-20, 50% glycerol.

 

10X nTaq-HOT buffer

Containing 15 mM MgCl2

 

Cautions

nTaq-Pure HOT (purified from insect cell) requires 10 minutes of initial denaturation to ensure effective activation of the enzyme.

 

Standard reaction conditions

- PCR mixture

10X nTaq-HOT buffer

μl

nTaq-Pure HOT (purified from insect cell) DNA Polymerasea (5 units/μl)

0.2 μl

dNTP Mixture (2 mM each)

μl

Template DNA(0.1~500 ng/μl)

μl

Primer 1 (5 pmole/μl)

μl

Primer 2 (5 pmole/μl)

μl

Sterile water

up to 20 μl

aAdd the PCR polymerase at the final step

bPlasmid DNA, 0.1 ng~30 ng; genomic DNA, 50 ng~500 ng

 

- PCR cycle

Step

Temperature

Time

Cycle

Initial denaturationa

95

10 min

1

Denaturation

95

30 sec

25~35

Annealingb

55~65

30~60 sec

Elongation

72

1 min/kb

Final elongation

72

5 min

1

When cycles are over, keep the reaction mixture at 4; may add 10 mM EDTA until use to prevent DNA degradation.

aAt least 10 min of initial denaturation time is required to fully activate the chemically modified PCR DNA polymerase.

bRecommended annealing temperatures is 5 to 10 below the lower Tm of the two primers used.

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