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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

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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01/22/2008 9:33:52 PM
Churchwolf responded with a statement of type: Agree
A very helpful and clear demonstration. Thanks!
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11/04/2008 7:26:12 AM
sj responded with a statement of type: Question

With this kit is it best to do an alcohol precipitation after or unnecessary?

 

Thank you

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11/04/2008 8:18:08 AM
QIAGEN Technical Service responded with a statement of type: Neutral

 

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

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07/18/2009 10:51:53 AM
ooisoonkeat responded with a statement of type: Neutral
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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07/29/2007

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Journal of Visualized Experiments (JoVE) is an online research journal employing visualization to increase reproducibility and transparency in biological sciences.

 

ISSN 1940-087X

 

 

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