Navigation Links
Basic Plasmid DNA Isolation Protocol

PLG can be used in small and large scale protocols based on alkaline lysis procedure.(1,2,3)

The basic protocol given here works with E. coli cultures grown for 1214 hours in up to 500 ml of amplified or non-amplified LB or DYT, or in 250 ml Terrific Broth.

  1. Pellet bacteria from the culture at 10,000 x g for 5 minutes at room temperature.
  2. Resuspend bacterial pellet in a total of 15 ml 25 mM Tris-Cl/10 mM EDTA (pH 8.0). Pipet up and down or vortex as necessary to fully resuspend the bacteria.
  3. Add 15 ml room temperature 0.2 N NaOH/1.0% SDS to the suspension. Mix thoroughly by repeated gentle inversion. Do not vortex.
  4. Add 15 ml ice-cold 7.5 M Ammonium Acetate to the lysate. Mix thoroughly by repeated gentle inversion. Do not vortex.
  5. Centrifuge at 15,500 x g for 30 minutes at 4C.
  6. Recover resulting supernatant and divide it between 2 pre-spun (1500 x g for 23 minutes) PLG 50 ml Heavy tubes.
  7. Add 20 ml Phenol-Chloroform-Isoamyl Alcohol (PCI, 25:24:1) to the sample in each PLG 50 ml Heavy tube and mix thoroughly by repeated inversion. Do not vortex. Centrifuge at 1500 x g for 5 minutes to separate the phases.
  8. Recover resultant aqueous phases to fresh pre-spun PLG 50 ml Heavy tubes and repeat step 7 above.
  9. Recover resultant aqueous phases to fresh pre-spun PLG 50 ml Heavy tubes. Add 20 ml Chloroform-Isoamyl Alcohol (CI, 24:1) and extract as in step 7 above.
  10. Recover resultant aqueous phases to suitable tubes and process or precipitate the samples as per your protocols and procedures.
References
  1. Emanuel, J.R. (1992) Phase Lock Gel plasmid micropreps: direct insert screening, probe synthesis and sequencing within one day. Nucleic Acids Res. 20:625.
  2. Birnboim, H.C. and Doly, J. (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl. Acids Res. 7:1513-1522.
  3. Murphy, N.R. and Hellwig, R. J. (1996) Improved nucleic acid organic extraction through use of a unique gel barrier matrix. BioTechniques 21:934-939.

'"/>

Source:


Page: All 1 2

Related biology technology :

1. Basic Plasmid DNA Isolation Protocol
2. Array Analysis: The Basics
3. Northern Analysis: The Basics
4. Nuclease Protection Assays: The Basics
5. Efficiently Insert Unique Restriction Sites into Plasmid Vectors
6. High-Efficiency Site-Directed Mutagenesis of Large Plasmid Vectors
7. Simple Purification of DNA from Plasmid Minipreps, PCR Amplifications and Agarose Gels
8. Generate High-Quality, Directional Plasmid cDNA Libraries
9. New Reporter Plasmids for Studying Interferon-Stimulated Signal Transduction Pathways
10. New Fusion Trans-Activator Plasmids for Studying Signal Transduction Pathways
11. Comparison of Growth Techniques and Media for the Purpose of Plasmid Isolation from E. coli Using the Eppendorf Perfectprep Plasmid Mini Kit
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:6/23/2016)... SILVER SPRING, Md. , June 23, 2016 ... evidence collected from the crime scene to track the criminal ... sick, and the U.S. Food and Drug Administration (FDA) uses ... Sound far-fetched? It,s not. ... whole genome sequencing to support investigations of foodborne illnesses. Put ...
(Date:6/23/2016)... 2016  The Biodesign Challenge (BDC), a university competition ... harness living systems and biotechnology, announced its winning teams ... New York City . The ... projects at MoMA,s Celeste Bartos Theater during the daylong ... senior curator of architecture and design, and Suzanne ...
(Date:6/23/2016)... ... , ... In a new case report published today in STEM CELLS Translational ... lymphedema after being treated for breast cancer benefitted from an injection of stem cells ... this debilitating, frequent side effect of cancer treatment. , Lymphedema refers to ...
(Date:6/23/2016)... YORK , June 23, 2016 ... trading session at 4,833.32, down 0.22%; the Dow Jones Industrial ... S&P 500 closed at 2,085.45, down 0.17%. Stock-Callers.com has initiated ... INFI ), Nektar Therapeutics (NASDAQ: NKTR ), ... Therapeutics Inc. (NASDAQ: BIND ). Learn more about ...
Breaking Biology Technology:
(Date:6/3/2016)... LONDON , June 3, 2016 /PRNewswire/ ... Transport Management) von Nepal ... ,Angebot und Lieferung hochsicherer geprägter Kennzeichen, einschließlich ... weltweit führend in der Produktion und Implementierung ... an der Ausschreibung im Januar teilgenommen, aber ...
(Date:6/2/2016)... June 2, 2016 Perimeter Surveillance ... Unmanned Systems, Physical Infrastructure, Support & Other Service  ... visiongain offers comprehensive analysis of the global ... will generate revenues of $17.98 billion in 2016. ... Inc, a leader in software and hardware technologies for ...
(Date:5/20/2016)... 20, 2016  VoiceIt is excited to announce ... By working together, VoiceIt and VoicePass ... and VoicePass take slightly different approaches to voice ... security and usability. ... new partnership. "This marketing and technology ...
Breaking Biology News(10 mins):