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EZ™ Random Primer DNA Labeling Kit

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1 EZ™ Random Primer DNA Labeling Kit EZ022S - 40 rxns ₩ 180,000 추가
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Product description

Random Primer DNA Labeling Kit is designed for labeling template DNA to possess high specific radioactivity. This kit utilizes 9 mer random primers and the Klenow DNA Polymerase (exo-) which lacks 3' → 5' exonuclease activity. Each reaction is begun by adding denatured DNA to a mixture of dNTP, Klenow DNA Polymerase (exo-). Labeled dNTP incorporate to complementary sequences on template DNA and serve as random primer 9mer for synthesis of complementary DNA strands by Klenow DNA Polymerase (exo-). Conventional protocols require reaction times of 30 min to 1 hour. Enzynomics kit is capable of generating DNA probes of high specific activity of 1x109 dpm/μg in as little as 2-5 minutes. DNA labeling by this kit can be used in Northern, Southern blot.

 

 

Figure 1. DNA labeling by the random primed method.

 

Kit components

- Control DNA (λDNA Hind III 25 ng/μl)

- Random primer (9mer, 100 pmol/μl)

- dATP (0.25 mM)

- dGTP (0.25 mM)

- dTTP (0.25 mM)

- dCTP (0.25 mM)

- Klenow DNA Polymerase, exo- (5 units/μl)

- 10X Klenow DNA Polymerase buffer

- Stop buffer (0.25 M EDTA, pH 8.0)

- Distilled sterilized water

* Store all components at -20

* Reagents and materials not provided: [α-32P] dCTP Solution (100 μCi)

 

Quick protocol 

1. Combine the following components and heat at 95 for 3 min and then cool on ice.

Component

Vol.

Final Conc.

Template

x μl

25 ng

Random primer (9mer)

2 μl

200 pmol

Distilled water

up to 14 μl

 

 

2. Add the following components and Incubate at 37 for 5 min.

Component

Vol.

Final Conc.

dATP

2 μl

20 μM

dGTP

2 μl

20 μM

dTTP

2 μl

20 μM

[α-32P]dCTP

x μl

50 μCi

Klenow 10X buffer

2.5 μl

1X

Klenow DNA Polymerase, exo-

1 μl

5 unit

Total reaction volume

25 μl

 

 

3.     Add 3 μl of stop buffer.

 

● Material Safety Data Sheet

● Quality Data

Figure 2. Time course of radionucleotide incorporation in a Enzynomics kit reaction using 25 ng λ DNA Hind III




Figure 3. Time course of radionucleotide incorporation compared with reaction of competitors




Figure 4. Each company protocol reaction mixture loaded onto 1 % agarose gel




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