NDLI: Preparation of (32)P-end-labeled DNA fragments for performing DNA-binding experiments.

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Preparation of (32)P-end-labeled DNA fragments for performing DNA-binding experiments.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Carey, Michael F. Peterson, Craig L. Smale, Stephen T.
Abstract	The generation of a uniquely (32)P-end-labeled DNA fragment is essential for DNA-binding experiments such as DNase I footprinting and ethylation interference. We describe here a protocol for end-labeling a restriction fragment. For a plasmid DNA bearing a region containing the binding site of interest, cleaving with a single restriction endonuclease generates a 5' overhang containing a phosphate. This is generally necessary for both common forms of fragment end-labeling: phosphorylation with polynucleotide kinase and 'filling in the end' with DNA polymerases (e.g., Klenow fragment). For the phosphorylation reaction, as described here, the phosphate is removed with calf intestinal phosphatase or bacterial alkaline phosphatase, and the resulting free 5'-OH is phosphorylated with polynucleotide kinase and [Î³-(32)P]ATP. This generates a plasmid labeled at each end with Î³-(32)P. The molar amount of plasmid DNA must be below the amount of ATP added to the reaction and the ATP must be of sufficiently high specific activity to generate a fragment labeled to the extent necessary for many DNA-binding experiments. To generate a uniquely end-labeled DNA fragment, the labeled plasmid is heat-treated to inactivate any remaining kinase and recleaved with a second endonuclease, releasing a short DNA fragment and a longer vector fragment. The DNA fragment is purified from the labeled vector on a 5%-8% native polyacrylamide gel. The preparation and labeling of DNA restriction fragments typically takes 1-2 d.
File Format	HTM / HTML
ISSN	19403402
Issue Number	5
Volume Number	2013
e-ISSN	15596095
Journal	Cold Spring Harbor Protocols
Language	English
Publisher	Cold Spring Harbor Laboratory Press
Publisher Date	2013-05-01
Publisher Place	United States
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Discipline Clinical Laboratory Techniques Dna Metabolism Phosphorus Radioisotopes Isolation & Purification Isotope Labeling Methods Phosphoric Monoester Hydrolases Phosphorylation Plasmids Polynucleotide 5'-hydroxyl-kinase Journal Article
Content Type	Text
Resource Type	Article
Subject	Biochemistry, Genetics and Molecular Biology

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