Purifiant l'ADN plasmidique des colonies bactériennes en utilisant le kit Qiagen Miniprep Kit

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

Purification d'ADN plasmidique de E. coli est une technique de base pour le clonage moléculaire. D'épuration à petite échelle (miniprep) de moins de 5 ml de culture bactérienne est un moyen rapide pour la vérification clone ou l'isolement de l'ADN, suivie par d'autres réactions enzymatiques (réaction en chaîne de la polymérase et la digestion des enzymes de restriction). Ici, nous enregistrée sur bande vidéo les procédures générales de miniprep à travers le QIAGEN Miniprep Kit QIAprep 8, visant à introduire cette technique très efficace pour les débutants en général des techniques de biologie moléculaire. La procédure entière est basée sur lyse alcaline des cellules E. coli suivie par adsorption de l'ADN sur de la silice en présence du sel. Il se compose de trois étapes: 1) la préparation et la compensation d'un lysat bactérien, 2) l'adsorption de l'ADN sur la membrane QIAprep, 3) le lavage et l'élution de l'ADN plasmidique. Toutes les étapes sont réalisées sans l'utilisation de phénol, du chloroforme, CsCl, le bromure d'éthidium, et sans précipitation à l'alcool. Il prend généralement moins de 2 heures pour terminer la procédure.

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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