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High Yield Miniprep Protocol

This document provides a high-yield protocol for purifying up to 30 micrograms of plasmid DNA from bacterial cultures grown to high cell density. The protocol uses the QIAprep Spin Miniprep Kit and involves lysing bacterial cells, binding DNA to a spin column, washing and then eluting the purified plasmid DNA.

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Adison Wong
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© Attribution Non-Commercial (BY-NC)
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Download as PDF, TXT or read online on Scribd
395 views

High Yield Miniprep Protocol

This document provides a high-yield protocol for purifying up to 30 micrograms of plasmid DNA from bacterial cultures grown to high cell density. The protocol uses the QIAprep Spin Miniprep Kit and involves lysing bacterial cells, binding DNA to a spin column, washing and then eluting the purified plasmid DNA.

Uploaded by

Adison Wong
Copyright
© Attribution Non-Commercial (BY-NC)
Available Formats
Download as PDF, TXT or read online on Scribd
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QIAprep Spin Miniprep Kit High-Yield Protocol

For purification of up to 30 g plasmid DNA


This protocol is for use with the QIAprep Spin Miniprep Kit (cat. nos. 27104 and 27106), and is specially optimized for high-yield purification of high-copy plasmid DNA from bacterial cultures grown to a high cell density (e.g., culture grown in 5 ml 2x YT medium). For further details, including safety information, refer to the QIAprep Miniprep Handbook. Note: Certain insertplasmid constructs may reduce the copy number of the plasmid, resulting in lower yields after preparation. In addition, not all E. coli strains are suitable for high-yield plasmid purification. This protocol was verified using E. coli DH5 carrying pCMV plasmid.

Figure 1. Higher plasmid DNA yields with cultures grown in 2 xYT medium. Plasmid DNA was isolated from E. coli DH5 (carrying plasmid pCMV) cultured in LB medium or 2x YT medium, using the QIAprep Spin Miniprep standard protocol and the high-yield protocol, respectively.

Notes before starting



Add LyseBlue reagent to Buffer P1 at a ratio of 1 to 1000. Add the provided RNase A solution to Buffer P1, mix, and store at 28C. Add ethanol (96100%) to Buffer PE before use (see bottle label for volume). All centrifugation steps, unless stated otherwise, are carried out at 13,000 rpm (~17,900 x g) in a conventional table-top microcentrifuge. Symbols: centrifuge processing; vacuum processing.

Procedure
1. Inoculate 5 ml 2x YT medium with the bacterial strain and incubate the culture for 1216 h at 37C. Avoid overgrowth, which leads to cell lysis and reduced plasmid yields.

Sample & Assay Technologies

2. 3. 4. 5. 6. 7.

Pellet 5 ml bacterial overnight culture by centrifugation at >8000 rpm (6800 x g) for 3 min at room temperature (1525C). Completely resuspend pelleted bacterial cells in 250 l Buffer P1 and transfer to a microcentrifuge tube. Ensure that no clumps of cells remain. Add 250 l Buffer P2 and mix by inverting the tube 1012 times until the solution turns completely blue. Incubate for 5 min. Do not allow lysis to proceed for more than 5 min. Add 350 l Buffer N3 and mix immediately and thoroughly by inverting the tube 1012 times until the solution turns colorless. Centrifuge for 10 min. Apply the supernatant from step 6 to the QIAprep spin column by decanting or pipetting. Centrifuge for 3060 s and discard the flow-through, or apply vacuum to the manifold to draw the solution through the QIAprep spin column and switch off the vacuum at source.

8. 9.

Wash the QIAprep spin column by adding 500 l Buffer PB. Centrifuge as in step 7, or apply vacuum to draw the solution through the QIAprep spin column as in step 7. Wash the QIAprep spin column by adding 750 l Buffer PE. Centrifuge as in step 7, or apply vacuum to draw the solution through the QIAprep spin column as in step 7. Transfer the QIAprep spin column to the collection tube.

10. 11.

Centrifuge for 1 min to remove residual wash buffer. Place the QIAprep spin column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 60 l Buffer EB (10 mM TrisCl, pH 8.5) to the center of the QIAprep spin column, let it stand for 1 min, and centrifuge for 1 min.

Ordering Information
Product QIAprep Spin Miniprep Kit (50) Contents For 50 high-purity plasmid minipreps: 50 QIAprep Spin Columns, Reagents, Buffers, Collection Tubes (2 ml) QIAprep Spin Miniprep Kit (250) For 250 high-purity plasmid minipreps: 250 QIAprep Spin Columns, Reagents, Buffers, Collection Tubes (2 ml) For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.qiagen.com or can be requested from QIAGEN Technical Services or your local distributor.
Trademarks: QIAGEN, QIAprep, LyseBlue (QIAGEN Group). 1070088 09/2011 2011 QIAGEN, all rights reserved.

Cat. no. 27104

27106

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