11585584001

High Prime DNA Labeling Kit

sufficient for 50 labeling reactions (0.01 to 2 μg DNA per assay)

• UNSPSC Code: 41105500

Properties

• usage: sufficient for 50 labeling reactions (0.01 to 2 μg DNA per assay)
• manufacturer/tradename: Roche
• storage temp.: −20°C

Description

General description

Sample Materials

• Either linear or supercoiled plasmid DNA, λDNA
• Shorter fragments of 200 bp
• DNA fragments in molten agarose
• <10 ng DNA

Assay Time: 20 minutes for a standard labeling assay

Note: The length of DNA to be labeled does not influence the reaction. Maximal incorporation is achieved after incubation of 30 to 60 minutes.

Convenient kit for the radioactive and nonradioactive labeling of DNA with modified deoxyribonucleoside triphosphate using random oligonucleotides as primers. In this method, the complementary DNA strand of denatured DNA is synthesized by Klenow Polymerase using the 3′-OH termini of the random oligonucleotides as primers.

Specificity

Heat inactivation: Stop the reaction by adding 2 μl 0.2 M EDTA (pH 8.0) and/or by heating to 65 °C for 10 minutes.

The standard assay routinely yields a specific activity of 2 x 10⁹ dpm/μg, using different substrate DNAs after 10 minutes of incubation.

Application

Based on the High Prime principle, the High Prime DNA Labeling Kit is used for rapid random-primed DNA labeling. The method enables the labeling of DNAs available only in minute quantities, for example, DNA restriction fragments isolated from gels or in molten agarose. The separately provided nucleotide solutions offer a choice in the selection of modified deoxyribonucleoside triphosphates (e.g., ³²P, ³⁵S, ³H, digoxigenin, fluorescein, biotin, or rhodamine). High Prime DNA Labeling Kit has been used for:

• high prime labeled probes are used in a variety of hybridization techniques: Southern blots[1]
• northern blots
• screening of gene libraries
• in situ hybridizations
• subtractive hybridization (SSH) assay

The High Prime DNA Labeling Kit offers the same characteristics as High Prime.

Packaging

1 kit containing 6 components.

Principle

The "random primed" DNA labeling method originally developed by Feinberg and Vogelstein is based on the hybridization of oligonucleotides of all possible sequences to the denatured DNA to be labeled.Input DNA serves is the only template for synthesis of labeled DNA, making it possible to label minimal amounts of DNA (10 ng) using this method. Complementary DNA strands are synthesized using Klenow polymerase at the 3′-OH termini of randomized oligonucleotides used as primers. Modified deoxyribonucleoside triphosphates (e.g., labeled with ³²P, ³⁵S, ³H, digoxigenin, biotin, fluorescein, or rhodamine) added to the reaction are readily incorporated into newly synthesized DNA strands.

Preparation Note

Working solution: dATP, dGTP, dTTP mixture:
For one labeling reaction pipette:
1 μl dATP, (vial 2)
1 μl dGTP, (vial 4)
1 μl dTTP, (vial 5)
to a reaction vial.

Note: If the same type of labeled deoxyribonucleoside-triphosphate is used repeatedly, we recommend the preparation of a mixture of equal parts of the other three deoxyribonucleoside-triphosphates for convenience.

Other Notes

For life science research only. Not for use in diagnostic procedures.

Kit Components Only

• High Prime Reaction Mixture (random primer mixture, Klenow polymerase, labeling grade, and 5x stabilized reaction buffer in 50% (v/v) glycerol) 5x concentrated
• dATP: 2′-Deoxyadenosine-5′-triphosphate in Tris buffer 0.5 mM
• dCTP: 2′-Deoxycytidine-5′-triphosphate in Tris buffer 0.5 mM
• dGTP: 2′-Deoxyguanosine-5′-triphosphate in Tris buffer 0.5 mM
• dTTP: 2′-Deoxy-thymidine-5′-triphosphate in Tris buffer 0.5 mM
• Control DNA: λDNA 12.5 μg/ml

Safety Information

• Storage Class: 12 - Non Combustible Liquids
• wgk_germany: WGK 1
• flash_point_f: does not flash
• flash_point_c: does not flash