Zuiveren Plasmide DNA van bacteriële kolonies Met behulp van de Qiagen Miniprep Kit


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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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Plasmide DNA-zuivering van E. coli is een kern-techniek voor moleculaire klonen. Kleinschalige zuivering (Miniprep) van minder dan 5 ml bacteriecultuur is een snelle manier voor het klonen verificatie of DNA-isolatie, gevolgd door verdere enzymatische reacties (polymerase kettingreactie en restrictie-enzym digestie). Hier hebben we video opgenomen van de algemene procedures van Miniprep door de QIAGEN's QIAprep 8 Miniprep Kit, gericht op de invoering van deze zeer efficiënte techniek om de algemene beginners voor moleculaire biologische technieken. De hele procedure is gebaseerd op alkaline lysis van E. coli-cellen gevolgd door adsorptie van DNA op silica in de aanwezigheid van hoge zout. Het bestaat uit drie stappen: 1) de voorbereiding en afwikkeling van een bacteriële lysaat, 2) adsorptie van DNA op het membraan QIAprep, 3) het wassen en elutie van het plasmide DNA. Alle stappen worden uitgevoerd zonder het gebruik van fenol, chloroform, CsCl, ethidiumbromide, en zonder alcohol neerslag. Het duurt meestal minder dan 2 uur tot het einde van de gehele procedure.



  1. A very helpful and clear demonstration. Thanks!

    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

    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

    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.

    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

    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.

    Posted by: Anonymous
    March 23, 2010 - 5:02 AM

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