Purificação de DNA plasmidial colônias bacterianas Usando o Kit Qiagen Miniprep

Biology

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Summary

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Zhang, S., Cahalan, M. D. Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit. J. Vis. Exp. (6), e247, doi:10.3791/247 (2007).

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Abstract

Purificação de DNA plasmidial de E. coli é uma técnica de núcleo para clonagem molecular. Purificação de pequena escala (miniprep) de menos de 5 ml de cultura bacteriana é uma maneira rápida para verificação clone ou o isolamento do DNA, seguida por outras reações enzimáticas (reação em cadeia da polimerase e digestão enzima de restrição). Aqui, vídeo-gravados os procedimentos gerais de miniprep através QIAprep a QIAGEN Kit Miniprep 8, com o objetivo de introduzir esta técnica altamente eficiente para os novatos em geral para técnicas de biologia molecular. Todo o procedimento é baseado em lise alcalina de células de E. coli seguido por adsorção de DNA em sílica na presença de sal. Ele consiste de três etapas: 1) preparação e limpeza de um lisado bacteriano, 2) adsorção de DNA para a membrana QIAprep, 3) lavagem e eluição do DNA plasmidial. Todas as etapas são realizadas sem o uso de fenol, clorofórmio, CsCl, brometo de etídio, e sem precipitação álcool. Ele normalmente leva menos de 2 horas para terminar todo o procedimento.

Comments

6 Comments

  1. A very helpful and clear demonstration. Thanks!

    Reply
    Posted by: Anonymous
    January 22, 2008 - 9:33 PM
  2. With this kit is it best to do an alcohol precipitation after or unnecessary?   Thank you

    Reply
    Posted by: Anonymous
    November 4, 2008 - 7:26 AM
  3.   Hello from QIAGEN, There is no need to do an alcohol precipitation after this procedure - the DNA is ready to use as eluted from the QIAprep column. You can see the handbook for this kit online here if you would like:     http://www1.qiagen.com/literature/handbooks/literature.aspx?id=1000²48 -QIAGEN Technical Service     http://www1.qiagen.com/Contact/QIAGENWorldWide.aspx

    Reply
    Posted by: Anonymous
    November 4, 2008 - 8:18 AM
  4. By using this kit, what can I do if I'd like to increase the yield of plasmid with a low copy number? What is the maximum volume of culture that can be harvested?
    Thank you very much.

    Reply
    Posted by: Soon Keat O.
    July 18, 2009 - 10:51 AM
  5. Your demonstration is very good but you didn't mentioned the volumes of buffer added and the speed and time of centrifuge

    Reply
    Posted by: Anonymous
    March 2, 2010 - 10:40 AM
  6. Thanks for doing that video. There's not a lot of theory in there, but it is surely helpful for beginners. I'm holding onto it for future coworkers.

    Reply
    Posted by: Anonymous
    March 23, 2010 - 5:02 AM

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