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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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Purifying Plasmid-DNA aus Bakterien-Kolonien mit dem Qiagen Miniprep Kit

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

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Cite this Article: Purifying Plasmid-DNA aus Bakterien-Kolonien mit dem 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: Purifying Plasmid-DNA aus Bakterien-Kolonien mit dem Qiagen Miniprep Kit

Plasmid DNA Aufreinigung aus E. coli ist ein Kern-Technik für die molekulare Klonen. Kleine Reinigung (Miniprep) von weniger als 5 ml Bakterienkultur ist ein schneller Weg für Klon Überprüfung oder DNA-Isolierung, durch weitere enzymatische Reaktionen (Polymerase-Kettenreaktion und Restriktionsverdau) gefolgt. Hier haben wir das allgemeine Verfahren der Miniprep durch die QIAGEN QIAprep 8 Miniprep Kit, mit dem Ziel der Einführung dieser hoch effizienten Technik den allgemeinen Anfänger für molekularbiologische Techniken auf Video aufgezeichnet. Das gesamte Verfahren ist auf alkalischen Lyse von E. coli Zellen, die durch Adsorption von DNA an Silica folgte in der Gegenwart von hohen Salz. Es besteht aus drei Schritten: 1) Vorbereitung und Abwicklung von einer bakteriellen Lysat, 2) Adsorption von DNA an die Membran QIAprep, 3) Waschen und Elution von Plasmid-DNA. Alle Schritte werden ohne den Einsatz von Phenol, Chloroform, CsCl, Ethidiumbromid, und ohne Alkohol-Fällung durchgeführt. Es dauert in der Regel weniger als 2 Stunden, um die gesamte Vorgang zu beenden.

Disclosures: Purifying Plasmid-DNA aus Bakterien-Kolonien mit dem Qiagen Miniprep Kit

Ask the Author: Purifying Plasmid-DNA aus Bakterien-Kolonien mit dem 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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