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DecaLabel™ DNA Labeling Kit

#K0621	for 10 reactions
#K0622	for 30 reactions
Related Documents (in pdf, ~30-50 KB): Detailed Protocol: #K0621, #K0622 MSDS (English) MSDS (English-USA) MSDS (German)

 

Features

• Radioactive labeling.
• Effective - high specific radioactivity of probes (>1x10⁹ dpm/µg DNA).
• Fast - 5 minutes at 37°C.
• Efficient priming of labeling reaction with random decamers.
• High yields with Klenow Fragment, exo-: no degradation of a labeled probe during reaction.
• Convenient - suitable for either radiolabeled dATP or dCTP.

Description
The DecaLabel™ DNA Labeling Kit is an advanced system for fast synthesis of radiolabeled DNA probes of high specific activity. The kit is based on the improved random-primed method developed by Feinberg and Vogelstein (1, 2).
The primary improvement over traditional random-prime kits involves the use of random decamers instead of hexamers to ensure more efficient annealing with DNA at 37°C. The use of Klenow Fragment, exo-, which has been genetically engineered to remove exonuclease activity, enhances the labeling. This improved enzyme does not degrade the labeled probe during the reaction, resulting in uniform labeling of even low amounts of DNA. DNA molecules of any size can be labeled with this kit.

Principle
Random decamers are annealed to a denatured template DNA, and radiolabeled dNTPs are then incorporated into new DNA strands by Klenow Fragment, exo- (see Fig.1 below).

Applications
Generation of radiolabeled DNA probes for use in a variety of hybridization experiments: Southern and Northern blots, colony hybridizations, dot/slot blots and in situ hybridizations.

Quality Control
The kit is tested in preparation of labeled lambda DNA/HindIII fragments. The typical specific radioactivity of the synthesized probe is >1x10⁹  dpm/µg DNA.

Components of the Kit

• Klenow Fragment, exo-
• Decanucleotide in Reaction Buffer
• Mix A (-dATP)
• Mix C (-dCTP)
• dNTP Mix
• Control Template
• Water, nuclease-free
• Detailed Protocol (in pdf, ~30 KB):
  • #K0621
  • #K0622

Figure 1. DNA labeling by random primer method.
* [alfa-³²P]-dNTP, [alfa-³²P]-dNTP or biotin-dUTP can be used.

Figure 2. Influence of reaction time and amount of template on specific activity of a DNA probe.
Varying amounts of lambda DNA/HindIII fragments were labeled with 50 µCi [alfa-³³P]-dCTP (2500 Ci/mmol) according to the protocol (in PDF format, 30 KB) supplied with the kit.

Related Products

Genomic DNA Purification Kit DNA Gel Extraction Kit SensiBlot™ Plus Nylon Membrane Agarase

References

1. Feinberg, A.P., Vogelstein, B., A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity, Biochem., 132, 6-13, 1983.
2. Feinberg, A.P., Vogelstein, B., A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity, Addendum, Biochem., 137, 266-267, 1984.

Updated balandžio 24, 2008 18:53