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Polymerases

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nTaq-HOT

No Product Name Cat.# Conc Size Price(₩) Add
1 nTaq-HOT P725 5 units/μl 250 units ₩ 72,000 추가
2 nTaq-HOT P750 5 units/μl 500 units ₩ 130,000 추가
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Product description

A gene encoding the Thermus aquaticus (Taq) DNA polymerase was cloned and expressed in E. coli, and the enzyme was purified to homogeneity. nTaq-HOT is a hot start PCR polymerase which remains inactive at temperatures lower than 75 as a result of the chemical modifications. Therefore, a heat activation step is required to activate nTaq-HOT. Activation is accomplished at denaturation steps of PCR cycles and, thus, nTaq-HOT is active only after initiation PCR cycles. 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-HOT, thus increasing the specificity and efficiency of target DNA amplification.

 

Characteristics

- 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 higher ordered 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)

- GC Melt I

- GC Melt II

- 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

 

GC Melt l and ll

- This product is useful for amplification of DNA with GCrich sequences or to avoid amplification of non-specific bands (Use of GC Melt may reduce PCR efficiency).

- In general, use 1X strength in PCR reaction by diluting the 10X solution, but the amount should be adjusted for optimal results.

 

Cautions

nTaq-HOT requires 10 minutes of initial denaturation to ensure effective activation of the enzyme.

 

Standard reaction conditions

- PCR mixturea

10X nTaq-HOT buffer

2 μl

nTaq-HOT DNA Polymerasea (5 units/μl)

0.2 μl

dNTP Mixture (2 mM each)

2 μl

Template DNAb (0.1~500 ng/μl)

1 μl

Primer 1 (5 pmole/μl)

1 μl

Primer 2 (5 pmole/μl)

1 μl

Sterile water

up to 20 μl

aAssemble the reaction mixture on ice

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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