使用Qiagen公司的小量制备试剂盒的细菌菌落纯化质粒DNA

Biology

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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

大肠杆菌质粒DNA纯化是分子克隆的核心技术。小规模小于5毫升细菌培养纯化(小量)为进一步酶促反应(聚合酶链反应和限制性内切酶消化),克隆验证或DNA分离的快捷方式。在这里,我们录像通过QIAGEN的QIAprep 8小量制备试剂盒小量的一般程序,旨在引进这种高效的技术,分子生物学技术的一般初学者。整个过程是基于碱裂解大肠杆菌细胞,在高盐存在的DNA吸附到硅。它包括三个步骤:)1,编制和结算的一种细菌裂解液,2)的DNA吸附到QIAprep膜,3)洗涤和质粒DNA洗脱。所有步骤都没有使用苯酚,氯仿,氯化铯,溴化乙锭,无酒精沉淀。它通常需要不到2个小时,以完成整个过程。

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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