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Polymerases

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2X TOPsimple™ DyeMIX–Forte

No Product Name Cat.# Conc Size Price(₩) Add
1 2X TOPsimple™ DyeMIX–Forte P510F 2X 1 ml ₩ 90,000 추가
2 2X TOPsimple™ DyeMIX–Forte P525F 2X 2.5 ml ₩ 180,000 추가
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Product description

2X TOPsimple™ DyeMIX-Forte is similarly formulated to the 2X TOPsimple™ PreMIX-Forte except that it contains loading dyes for further convenience of use. Thus, the reaction mixtures after PCR cycles are ready for agarose gel electrophoresis. This product includes nPfu-Forte (Cat. P410, P425), and is optimized to amplify high-fidelity and long DNA up to 10 kb.

 

Characteristics

- 2-Dye system: Easy for gel electrophoresis (xylene cyanol and Orange G)

- Blunt end product

 

Applications

- High fidelity DNA amplification for cloning

- Amplification of long DNA fragments (<10 kb)

- DNA amplification to produce blunt end products

- Site-directed mutagenesis

- Colony PCR

 

Components (2X)

nPfu-Forte DNA polymerase: 0.2 unit/μl

nPfu-Forte buffer (containing 4 mM MgSO4)

- dNTP mixture: 0.4 mM each

- Stabilizer

- Dye: Xylene cyanol and Orange G

 

Migration of dyes in agarose gel

In an ordinary agarose gel, xylene cyanol co-migrate with 4-kb DNA fragments, and Orange G with 50-bp DNA fragments.

 

Storage

table up to 18 months at -20 or 3 months at 4 (Storage at -20 is recommended).

 

Standard reaction conditions

- PCR mixturea

2X TOPsimple™ DyeMIX-Forte

10 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

aAssmeble the reaction mixture on ice

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

 

- PCR cycles

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.

 

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