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:5/16/2017)... May 16, 2017 Telehealth has long ... and something that has been kept completely separate ... But according to   Logicalis Healthcare Solutions , ... IT solutions and managed services provider ( www.us.logicalis.com ... overlooked – interrelationship between telehealth, imaging, and EHR ...
(Date:5/15/2017)... Somerset, N.J. (PRWEB) , ... May 15, 2017 ... ... advanced delivery technologies and development solutions for drugs, biologics and consumer health products, ... biotechnology company focused on rare genetic diseases, to support preclinical and clinical development ...
(Date:5/12/2017)... ARBOR, Mich. , May 12, 2017 ... selected to present at the 36th annual Michigan Growth ... Forum. GreenMark, a Delaware corporation ... be presenting to investors in attendance, including more than ... deliver health benefits to society through biobased targeting technologies. ...
(Date:5/11/2017)... (PRWEB) , ... May 11, 2017 , ... ... rodent neurons both are excellent resources for disease modeling and drug screening. Human ... differentiated into mature neurons for various applications, however, these often contain mixed population ...
Breaking Biology Technology:
(Date:4/5/2017)... KEY FINDINGS The global market ... CAGR of 25.76% during the forecast period of 2017-2025. ... for the growth of the stem cell market. ... MARKET INSIGHTS The global stem cell market is segmented ... The stem cell market of the product is segmented ...
(Date:3/30/2017)... The research team of The Hong Kong Polytechnic University (PolyU) ... ground breaking 3D fingerprint minutiae recovery and matching technology, pushing contactless ... use in identification, crime investigation, immigration control, security of access and ... ... A research team led by Dr Ajay Kumar ...
(Date:3/27/2017)... , March 27, 2017  Catholic Health Services ... Management Systems Society (HIMSS) Analytics for achieving Stage ... Model sm . In addition, CHS previously earned ... hospitals using an electronic medical record (EMR). ... high level of EMR usage in an outpatient ...
Breaking Biology News(10 mins):