Purificazione del DNA plasmidi da colonie batteriche Utilizzando il 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

Purificazione del DNA plasmidico da E. coli è una tecnica di base per la clonazione molecolare. Purificazione su piccola scala (miniprep) da meno di 5 ml di coltura batterica è un modo rapido per la verifica clone o isolamento del DNA, seguita da ulteriori reazioni enzimatiche (reazione a catena della polimerasi e la digestione enzimatica di restrizione). Qui, il video-registrato le procedure generali di miniprep attraverso il QIAGEN QIAprep 8 Kit Miniprep, con l'obiettivo di introdurre questa tecnica altamente efficiente per i principianti generale di tecniche di biologia molecolare. L'intera procedura si basa sulla lisi alcalina delle cellule di E. coli seguito da adsorbimento del DNA su silice in presenza di sale. Si compone di tre fasi: 1) la preparazione e la rimozione di un lisato batterico, 2) l'adsorbimento del DNA sulla membrana QIAprep, 3) lavaggio e l'eluizione del DNA plasmidico. Tutti i passaggi vengono eseguiti senza l'utilizzo di fenolo, cloroformio, CsCl, bromuro di etidio, senza precipitazione e alcool. Di solito ci vogliono meno di 2 ore per completare l'intera procedura.

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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