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Polymerases

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nTaq-Tenuto

No Product Name Cat.# Conc Size Price(₩) Add
1 nTaq-Tenuto (Mg2+ plus buffer) P225A 5 units/μl 250 units ₩ 84,000 추가
2 nTaq-Tenuto (Mg2+ free buffer) P225B 5 units/μl 250 units ₩ 84,000 추가
3 nTaq-Tenuto (Mg2+ plus buffer) P250A 5 units/μl 500 units ₩ 138,000 추가
4 nTaq-Tenuto (Mg2+ free buffer) P250B 5 units/μl 500 units ₩ 138,000 추가
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Product description

nTaq-Tenuto is nTaq (Cat. P025, P050) supplemented with 3'->5’ proofreading activity and a PCR enhancing factor for improved efficiency and fidelity. nTaq-Tenuto can be used to amplify DNA longer than 10 kb, which is difficult with common Taq polymerases alone. Thus, this product is improved in both fidelity (> 2 fold) of PCR products and amplification efficiency of longer PCR products.

 

Characteristics

- Molecular weight: 94 kDa

- Error rate: 3.0 X 10-6

- Thermal stability: Half life of 40 min at 95

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

 

Applications

- Amplification of long DNA fragments (<10 kb)

- Amplification of high-complexity template DNA

- Primer extension

- Colony PCR

- Labeling of DNA fragments with radioactive-isotopes

- Nucleotide sequencing

 

Supplied with

nTaq-Tenuto (Mg2+ plus buffer)

- 10X nTaq-Tenuto buffer (Mg2+ plus)

- dNTP Mixture (2 mM each)

- GC Melt I

- GC Melt II

- Sterile water

 

nTaq-Tenuto (Mg2+ free buffer)

- 10X nTaq-Tenuto buffer (Mg2+ free)

- dNTP Mixture (2 mM each)

- GC Melt I

- GC Melt II

- 25 mM MgCl2

- 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, 1 mM DTT, 0.5% NP-40, 0.5% Tween-20, 50% glycerol.

 

10X nTaq-Tenuto buffer

Mg2+ plus buffer: Containing 20 mM MgCl2

Mg2+ free buffer: MgCl2-free

 

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.

 

Standard reaction conditions

- PCR mixturea

10X nTaq-Tenuto buffer

2 μl

nTaq-Tenuto DNA Polymeraseb (5 units/μl)

0.2 μl

dNTP Mixture (2 mM each)

2 μl

Template DNAc (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

bAdd the PCR polymerase at the final step

cPlasmid DNA, 0.1 ng-30 ng; genomic DNA, 50 ng-500 ng

 

- PCR cycle

Step

Temperature

Time

Cycle

Initial denaturation

95

2 min

1

Denaturation

95

30 sec

25~35

Annealinga

55~65

30~60 sec

Elongation

72

1 min/kb

Final elongation

72

5 min

1

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

aRecommend annealing temperature is 5 to 10 below the lower Tm of the two primers used.



   
Figure 1. PCR amplification of long DNA fragments using nTaq-Tenuto.

DNA fragments up to 20 kb can be efficiently amplified by nTaq-Tenuto (Cat.# P225 or P250). Target DNAs with varying lengths were amplified by the use of following PCR cycles; 95℃ 2 min, (95℃ 30 sec, 55℃ 30 sec, 72℃ 1 min/kb) x 30, 72℃ 5 min. Note that elongation time increases by 1 min per kb.
Lane 1, 0.5 kb; lane 2, 1 kb; lane 3, 2 kb; lane 4, 3 kb; lane 5, 4.5 kb; lane 6, 8 kb; lane 7, 11 kb; lane 8, 20 kb; lane 9, 1 kb (+) Ladder Marker (Cat.# DM003)


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