Navigation Links
RNA-free Plasmid DNA Isolation Protocol

The following protocol is for use with 1 liter cultures grown in LB medium for 1214 hours. It is also used for cultures grown for no longer than 1214 hours in 0.5 liter of enriched growth media such as Terrific Broth (TB). The reagents used in the cleared lysate preparation (step 15) should be scaled down proportionately for smaller culture volumes down to 200 ml.

  1. Pellet bacteria from the culture at 10,000 x g for 5 minutes at 4C.
  2. Resuspend bacterial pellet in a total of 30 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 30 ml room temperature 0.2 N NaOH/1.0% SDS to the suspension. Mix thoroughly by repeated gentle inversion. Do not vortex.
  4. Add 30 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 resultant supernatant to a fresh centrifuge bottle. Do not carry over any whitish-grey pellet material.
  7. Add 54 ml (0.6 x volume) room temperature 100% Isopropanol to the supernatant. Mix thoroughly by repeated inversion. Do not vortex.
  8. Centrifuge at 15,500 x g for 30 minutes at 20C. Discard resultant supernatant.
  9. Add 25 ml 70% Ethanol to the pellet. Mix by repeated inversion and centrifuge at 15,500 x g for 5 minutes to re-pellet DNA.
  10. Discard resultant supernatant, carefully aspirate any excess Ethanol, and dry the pellet for 1530 mi nutes.
  11. Add 1.5 ml TE, (10 mM Tris-Cl, 1 mM EDTA, pH 8.0) containing 50 g/ml RNase A and 2.5 g/ml RNase T1 to the pellet and mix gently to dissolve pellet.
  12. Centrifuge for 12 minutes at 10,000 x g to consolidate sample, then transfer sample to a pre-spun (1500 x g for 12 minutes) PLG 15 ml Light tube.
    Note: 510 l of this suspension can be analyzed by 1% agarose gel electrophoresis to confirm that plasmid DNA is present.
  13. Incubate for 15 minutes at 37C in a water bath.
  14. Extract sample once with 2.0 ml PCI (Phenol-Chloroform-lsoamyl Alcohol 25:24:1). Thoroughly mix the aqueous and organic phases by repeated inversion. Do not vortex. Centrifuge at 1500 x g for 5 minutes to separate the phases.
  15. Add 2.0 ml PCI to the aqueous sample in the same PLG 15 ml tube and extract as in step 14.
  16. Add 2.0 ml Chloroform-Isoamyl Alcohol (24:1) to the aqueous sample in the same PLG 15 ml tube and extract as in step 14.
  17. Carefully transfer resultant aqueous phase (1.41.5 ml) to a suitable fresh tube and process or precipitate the sample as per your protocols and procedures.


'"/>

Source:


Page: All 1 2

Related biology technology :

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


(Date:6/23/2016)... , June 23, 2016  Blueprint Bio, a company ... to the medical community, has closed its Series A ... Nunez . "We have received a commitment ... capital we need to meet our current goals," stated ... us the runway to complete validation on the current ...
(Date:6/23/2016)... Francisco, CA (PRWEB) , ... June 23, 2016 ... ... (EDC) software, is exhibiting at the Pennsylvania Convention Center and will showcase its ... Annual conference. ClinCapture will also be presenting a scientific poster on Disrupting Clinical ...
(Date:6/23/2016)... , June 22, 2016  Amgen (NASDAQ: ... of the QB3@953 life sciences incubator to ... health. The shared laboratory space at QB3@953 was created ... a key obstacle for many early stage organizations - ... of the sponsorship, Amgen launched two "Amgen Golden Ticket" ...
(Date:6/22/2016)... 22, 2016 Research and Markets has announced ... report to their offering. ... from $29.3 billion in 2013. The market is expected to grow ... 2015 to 2020, increasing from $50.6 billion in 2015 to $96.6 ... during the forecast period (2015 to 2020) are discussed. As well, ...
Breaking Biology Technology:
(Date:5/20/2016)... , May 20, 2016  VoiceIt is ... partnership with VoicePass. By working together, ... experience.  Because VoiceIt and VoicePass take slightly different ... engines increases both security and usability. ... excitement about this new partnership. "This ...
(Date:5/12/2016)... 2016 WearablesResearch.com , a brand of ... results from the Q1 wave of its quarterly wearables ... consumers, receptivity to a program where they would receive ... insurance company. "We were surprised to see ... Michael LaColla , CEO of Troubadour Research, "primarily because ...
(Date:5/3/2016)...  Neurotechnology, a provider of high-precision biometric identification ... Identification System (ABIS) , a complete system for ... can process multiple complex biometric transactions with high ... face or iris biometrics. It leverages the core ... MegaMatcher Accelerator , which have been used in ...
Breaking Biology News(10 mins):