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

Biology

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Summary

Cite this Article

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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

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。

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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