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Purificação de DNA plasmidial colônias bacterianas Usando o Kit Qiagen Miniprep

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

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Cite this Article: Purificação de DNA plasmidial colônias bacterianas Usando o Kit Qiagen Miniprep

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: Purificação de DNA plasmidial colônias bacterianas Usando o Kit Qiagen Miniprep

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.

Disclosures: Purificação de DNA plasmidial colônias bacterianas Usando o Kit Qiagen Miniprep

Ask the Author: Purificação de DNA plasmidial colônias bacterianas Usando o Kit Qiagen Miniprep

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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