컨텐츠 바로가기 영역
주메뉴로 바로가기
본문으로 바로가기

All products

Home > PRODUCTS > All products

nPfu-Forte

No Product Name Cat.# Conc Size Price(₩) Add
1 nPfu-Forte P410 2.5 units/μl 100 units ₩ 75,000 추가
2 nPfu-Forte P425 2.5 units/μl 250 units ₩ 150,000 추가
Product Name Cat.# Size Price(₩) EA Total(₩) 삭제
Total : 장바구니에 담기 바로 구매 하기
plus minus 삭제

Product description

nPfu-Forte is Pfu DNA Polymerase supplemented with an enhancer, which maintains the activity of Pfu DNA Polymerase in the presence of inhibitors produced during high-temperature PCR cycles. Thus, high-fidelity DNA polymerization is obtained with nPfu-Forte, allowing amplification of DNA up to 10 kb long. nPfu-Forte is more effective than nPfu and has a high amplification rate (1 kb/min). Higher levels of PCR products are often obtained even with lowabundance template DNA.

 

Characteristics

- Molecular weight: 90 kDa

- Error rate: 2.8 X 10-7

- Thermal stability: Half life of 4 hr at 95

- Blunt end products

 

Applications

- High fidelity DNA amplification for cloning

- Amplification of long DNA fragments (<10 kb)

- DNA amplification to generate blunt end products

- Site-directed mutagenesis

- DNA sequencing

 

Supplied with

- 10X nPfu-Forte 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 30min 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

50 mM Tris-HCl (pH 8.2), 0.1 mM EDTA, 1 mM DTT,0.1% NP-40, 0.1% Tween-20, 50% glycerol.

 

10X nPfu-Forte buffer

Containing 20 mM MgSO4

 

Standard reaction conditions

- PCR mixturea

10X nPfu-Forte buffer

2 μl

nPfu-Forte DNA Polymeraseb (2.5 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

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 mixture at 4; may add 10 mM EDTA until use to prevent DNA degradation.

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

 


   

Figure 1. High-fidelity PCR amplification of DNA using nPfu-Forte.

DNA fragments up to 10 kb can be efficiently amplified by highfidelity nPfu-Forte (Cat.# P410 or P425). Target DNAs with varying lengths were amplified by 2.5 units of nPfu-Forte with the following PCR cycles; 95 2min, (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, 1 kb (+) DNA Ladder Marker (Cat.# DM003); lane 2 , 0.5kb; lane 3, 0.9 kb; lane 4, 2 kb; lane 5, 3 kb; lane 6, 5 kb; lane 7, 8 kb

List