Mapping et Bal 31

[invitefeecbae9]

Bonjour a tous,

Voila j'aimerais faire un mapping en utuilisant Bal 31 mais je trouve tres peu voire pas du tout de documentation sur un eventuel protocole.
Si quelqu'un pouvait m'aider ca serait tres gentil.

Merci
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[Flyingbike]

j'ai trouvé ça a partir de New England Biolabs

Nucleic Acids Res. 1978 May;5(5):1445-64.
Extracellular nucleases of pseudomonas BAL 31. III. Use of the double-strand deoxyriboexonuclease activity as the basis of a convenient method for the mapping of fragments of DNA produced by cleavage with restriction enzymes.
Legerski RJ, Hodnett JL, Gray HB Jr.

Abstract
We have previously characterized an extracellular nuclease from Pseudomonas BAL 31 which, in addition to other activities, displays a double-strand exonuclease activity which progressively shortens both strands of linear duplex DNA molecules from both termini. This degradation is accomplished without the introduction of detectable scissions away from the ends of the duplexes. When this nuclease is used to produce a series of progressively shortened samples from a linear duplex DNA, subsequent digestion of these samples with a site-specific restriction endonuclease and analysis of the resulting fragments by gel electrophoresis permits the rapid establishment of the order of the restriction enzyme fragments through the entire genome. This is accomplished by noting from the electropherograms the order in which the various restriction enzyme fragments become noticeably shortened or disappear. Using this method, the five cleavage sites for the endonuclease Hpa I and the single cleavage sites for the nucleases Hpa II and Pst I have been mapped in PM2 bacteriophage DNA. In a more stringent test of the method, 18 of the 24 fragments produced by cleavage of coliphage lambdab2b5c DNA with the Pst I nuclease have been mapped, and five of the six remaining fragments have been assigned to small regions of the genome.

PMID: 307230 [PubMed - indexed for MEDLINE]PMCID: PMC342095Free PMC Article

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[invitefeecbae9]

Merci beaucoup!!

[invitefeecbae9]

Et est ce que quelqu'un aurait deja utilise cette methode pour un mapping et pourrait me conseiller?

[A voir en vidéo sur Futura]

[invitefeecbae9]

En fait encore une question : Comment trouver la concentration adequate de l'enzyme sachant que je que Bal 31 coupe 750 bp par minute et que j'ai une concentration d'ADN de 2microgrammes par microlitre. Desole mais j'arrive vraiment pas a trouver des infos utiles.

Merci encore