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.
  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.



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:

(Date:12/1/2015)... ... December 01, 2015 , ... Matthew “Tex” VerMilyea, PhD, HCLD, has joined Texas ... oversee all IVF lab procedures as well as continue his research efforts into the ... miles to Auckland, New Zealand to bring home a High Complexity Clinical Laboratory Director ...
(Date:11/30/2015)... ... ... announced the opening of a new core patient care hub with the opening of ... are part of GSCG’s expansion efforts in Latin America. , Both the Arica and ... from around the world. , The clinics will be headed by Victor Perez, M.D. and ...
(Date:11/30/2015)... HOLLISTON, Mass. , Nov. 30, 2015 /PRNewswire/ ... HART ), a biotechnology company developing bioengineered ... has received written notification from The NASDAQ Stock ... minimum bid price requirements. The letter noted that ... of HART,s common stock having exceeded $1.00 per ...
(Date:11/30/2015)... Human Longevity, Inc. (HLI), the genomics-based, technology-driven ... Genomics, Inc., a leading genome informatics company offering highly ... The San Diego -based company has ... and Co-founder, Ashley Van Zeeland , Ph.D., who is ... of the deal were not disclosed. ...
Breaking Biology Technology:
(Date:11/20/2015)... 2015 NXTD ) ("NXT-ID" or ... growing mobile commerce market and creator of the Wocket® ... was recently interviewed on The RedChip Money Report ... weekend on Bloomberg Europe , Bloomberg Asia, Bloomberg ... NXTD ) ("NXT-ID" or the "Company"), a biometric ...
(Date:11/18/2015)... , Nov. 18, 2015  As new scientific discoveries ... doctors and other healthcare providers face challenges in better ... and patients. In addition, as more children continue to ... patient,s adulthood and old age. John M. ... Children,s Hospital of Philadelphia (CHOP) . --> ...
(Date:11/17/2015)... Paris , qui ... Paris , qui s,est tenu du ... leader de l,innovation biométrique, a inventé le premier scanner ... sur la même surface de balayage. Jusqu,ici, deux scanners ... les empreintes digitales. Désormais, un seul scanner est en ...
Breaking Biology News(10 mins):