Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit
Shenyuan Zhang, Michael D. Cahalan Department of Physiology and Biophysics, University of California, Irvine
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0:07 Introduction7 0:21 Subscription Lock21 0:46 Growing Up Bacteria46 1:24 Resuspending and Lysing Bacteria with Buffers P1 and P284 3:56 Precipitating Bacterial Protein with Buffer N3236 4:38 Assembling the Qiavac Manifold during Centrifugation278 5:15 Binding Plasmid DNA to Qiastrip and Washing with Buffers PB and PE315 7:19 Eluting DNA in Collection Tubes439 8:48 Conclusion528
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Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial culture is a quick way for clone verification or DNA isolation, followed by further enzymatic reactions (polymerase chain reaction and restriction enzyme digestion). Here, we video-recorded the general procedures of miniprep through the QIAGEN's QIAprep 8 Miniprep Kit, aiming to introducing this highly efficient technique to the general beginners for molecular biology techniques. The whole procedure is based on alkaline lysis of E. coli cells followed by adsorption of DNA onto silica in the presence of high salt. It consists of three steps: 1) preparation and clearing of a bacterial lysate, 2) adsorption of DNA onto the QIAprep membrane, 3) washing and elution of plasmid DNA. All steps are performed without the use of phenol, chloroform, CsCl, ethidium bromide, and without alcohol precipitation. It usually takes less than 2 hours to finish the entire procedure.
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Zhang S, Cahalan MD.. (2007). Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit. JoVE. 6. http://www.jove.com/index/details.stp?id=247, doi: 10.3791/247
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Allowed tags: i, b, u, sup, sub 
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| | 01/22/2008 9:33:52 PM
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Churchwolf responded with a statement of type: Agree
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A very helpful and clear demonstration. Thanks!
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| | 11/04/2008 7:26:12 AM
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sj responded with a statement of type: Question
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With this kit is it best to do an alcohol precipitation after or unnecessary?
Thank you
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| | 07/18/2009 10:51:53 AM
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ooisoonkeat responded with a statement of type: Neutral
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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.
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| | 03/02/2010 10:40:55 AM
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idrissa responded with a statement of type: Neutral
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Your demonstration is very good but you didn't mentioned the volumes of buffer added and the speed and time of centrifuge
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| | 03/23/2010 5:02:30 AM
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private responded with a statement of type: Neutral
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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.
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Mailer.URL: http://www.jove.com/index/details.stp?id=247 |
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