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The Journal of Visualized Experiments (JoVE) is a peer reviewed, PubMed-indexed video journal. Our mission is to increase the productivity of scientific research.

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

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Department of Physiology and Biophysics, University of California, Irvine (UCI)

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Cite this Article: Purifiant l'ADN plasmidique des colonies bactériennes en utilisant le kit Qiagen Miniprep Kit

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).

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

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.

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

Ask the Author: Purifiant l'ADN plasmidique des colonies bactériennes en utilisant le kit Qiagen Miniprep Kit

5 Comments

A very helpful and clear demonstration. Thanks!

1

Reply

Posted by: AnonymousJanuary 22, 2008, 9:33 PM

With this kit is it best to do an alcohol precipitation after or unnecessary?

 

Thank you

2

Reply

Posted by: sjNovember 4, 2008, 7:26 AM

 

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

2.1

Reply

Posted by: QIAGEN Technical ServiceNovember 4, 2008, 8:18 AM

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.

3

Reply

Posted by: Soon Keat O.July 18, 2009, 10:51 AM

Your demonstration is very good but you didn't mentioned the volumes of buffer added and the speed and time of centrifuge

4

Reply

Posted by: idrissaMarch 2, 2010, 10:40 AM

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.

5

Reply

Posted by: privateMarch 23, 2010, 5:02 AM

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