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キアゲンミニプレップキットを用いて細菌のコロニーからプラスミドDNAを精製

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

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Cite this Article: キアゲンミニプレップキットを用いて細菌のコロニーからプラスミドDNAを精製

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: キアゲンミニプレップキットを用いて細菌のコロニーからプラスミドDNAを精製

大腸菌からのプラスミドDNAの精製は、分子クローニングのためのコア技術です。細菌培養の100分の5以下mlの小規模の精製(ミニプレップ)は、さらに酵素反応(ポリメラーゼ連鎖反応および制限酵素消化)に続くクローンの検証またはDNAの分離のための手っ取り早い方法です。ここで、我々は分子生物学的手法の一般的な初心者にはこの非常に効率的な手法を導入することを目指し、QIAGENのQIAprep 8ミニプレップキットを介してミニプレップの一般的な手順をビデオ記録した。全体の手順は、高い塩の存在下でシリカにDNAの吸着に続いて大腸菌細胞のアルカリ溶解に基づいています。 1)QIAprep膜、3)洗浄し、プラスミドDNAの溶出にDNAの吸着細菌溶解物、2)の準備とクリア:それは3つのステップで構成されています。すべてのステップは、フェノール、クロロホルム、塩化セシウム、臭化エチジウムを使用せずに行われ、アルコール沈殿せずにされています。それは通常、この手順全体を完了するために2時間未満かかります。

Disclosures: キアゲンミニプレップキットを用いて細菌のコロニーからプラスミドDNAを精製

Ask the Author: キアゲンミニプレップキットを用いて細菌のコロニーからプラスミドDNAを精製

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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