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 minutes.
  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:
TAG: RNA free Plasmid DNA Isolation Protocol

(Date:10/22/2014)... North American crystal oscillator market report defines and segments ... of revenue. This market was valued at $531.4 million ... by 2018, at a CAGR of 3.3% from 2013 ... North American crystal oscillator market report, to get an ... a glimpse of the segmentation of the market, and ...
(Date:10/22/2014)... Currently offering a comprehensive portfolio ... Weighing, Rice Lake, and Ohaus, Pipette.com has added ... Sartorius CPA Semi-Micro Balance is ideal for lab ... user-friendly laboratory balance. , Sartorius is a global ... balances are well known for their high performance, ...
(Date:10/22/2014)... ROCKVILLE, Md. , Oct. 22, 2014   ... company developing novel pathogen-specific therapies for serious infections and ... Office has issued a Notice of Allowance for a ... product in its C. difficile program, SYN-004. This ... to SYN-004 in the U.S. and adds to the ...
(Date:10/22/2014)... HONG KONG , Oct. 22, 2014 ... enterprise, announced today that rare disease expert John ... president, research. Dr. McKew brings more than two decades ... at the National Institutes of Health, Wyeth Research and ... McKew will lead aTyr,s efforts to expand and translate ...
Breaking Biology Technology:The North American crystal oscillator market is expected to reach $623.6 million by 2018 - New Report by MicroMarket Monitor 2The North American crystal oscillator market is expected to reach $623.6 million by 2018 - New Report by MicroMarket Monitor 3The North American crystal oscillator market is expected to reach $623.6 million by 2018 - New Report by MicroMarket Monitor 4Pipette.com Launches the Addition of Sartorius CPA Semi-Micro Balance to Their Product Portfolio with Special Promotional Pricing 2Pipette.com Launches the Addition of Sartorius CPA Semi-Micro Balance to Their Product Portfolio with Special Promotional Pricing 3Synthetic Biologics Announces Allowance of Key U.S. Composition of Matter Patent for C. difficile Program 2Synthetic Biologics Announces Allowance of Key U.S. Composition of Matter Patent for C. difficile Program 3Synthetic Biologics Announces Allowance of Key U.S. Composition of Matter Patent for C. difficile Program 4aTyr Pharma Appoints John C. McKew, Ph.D., as Vice President, Research 2aTyr Pharma Appoints John C. McKew, Ph.D., as Vice President, Research 3
... DIEGO, Jan . 8 Arena Pharmaceuticals, Inc. (Nasdaq: ... to present at the 27th Annual J.P. Morgan Healthcare Conference ... p.m. Eastern Time) at the Westin St. Francis Hotel in ... is scheduled to provide an overview of the company, including ...
... 8 Palomar Medical Technologies, Inc. (Nasdaq: ... of light-based systems for cosmetic treatments, today announced ... the Swedish company Q-MED AB (OMX Nordic Exchange, ... based in Uppsala, Sweden. The international distribution ...
... SAN DIEGO, Jan. 7 Volcano Corporation,(Nasdaq: ... sales of,products for the diagnosis and treatment of coronary ... its previously announced acquisition of,Axsun Technologies, Inc. , ... manufacturer of lasers and optical,engines used in medical Optical ...
Cached Biology Technology:Arena Pharmaceuticals to Present at the 27th Annual J.P. Morgan Healthcare Conference 2Arena Pharmaceuticals to Present at the 27th Annual J.P. Morgan Healthcare Conference 3Palomar and Q-MED AB Terminate International Distribution Agreement 2Palomar and Q-MED AB Terminate International Distribution Agreement 3Palomar and Q-MED AB Terminate International Distribution Agreement 4Palomar and Q-MED AB Terminate International Distribution Agreement 5Volcano Announces Closing of Axsun Technologies Acquisition 2
(Date:10/22/2014)... Inc. (NASDAQ: AWRE ), a leading supplier of ... third quarter ended September 30, 2014. Revenue for ... of 40% compared to $4.3 million in the same quarter ... was $4.1 million compared to $1.0 million in the third ... month period was primarily due to: i) a $1.0 million ...
(Date:10/18/2014)... genetic conditions, a certain type of exome sequencing method ... traditional molecular diagnostic methods, according to a study appearing ... to coincide with the American Society of Human Genetics ... region of the genome (the complete set of genes ... has been rapidly applied in research settings and recent ...
(Date:10/17/2014)... available in German . ... few drugs. When treating overdoses, doctors are often limited to ... if there is a combination of drugs involved. So what ... swallows his grandmother,s pills? ETH professor Jean-Christophe Leroux from the ... an answer to this question. "The task was to develop ...
Breaking Biology News(10 mins):Aware, Inc. Reports Third Quarter 2014 Financial Results 2Aware, Inc. Reports Third Quarter 2014 Financial Results 3Aware, Inc. Reports Third Quarter 2014 Financial Results 4Aware, Inc. Reports Third Quarter 2014 Financial Results 5Aware, Inc. Reports Third Quarter 2014 Financial Results 6Aware, Inc. Reports Third Quarter 2014 Financial Results 7Study examines type of exome sequencing and molecular diagnostic yield 2Study examines type of exome sequencing and molecular diagnostic yield 3Emergency aid for overdoses 2Emergency aid for overdoses 3
... better understanding of brain injury, a way to rejuvenate ... monitor their glaucoma at home, number among this years ... What surprises me every time I walk through ... involving so many NJIT graduate and undergraduate students, said ...
... French . , A new brain-imaging study by ... ghrelin - a stomach hormone, acts on specific regions of ... and eating for pleasure. This study, published in the ... understanding and treating obesity, a condition affecting millions world-wide. ...
... The potential of no-tillage (NT) soils for increasing the soil ... Most of the previous studies about SOC accrual in NT ... depth), and not for the whole soil profile. The lack ... to conclusively ascertain the effects of NT farming on SOC ...
Cached Biology News:NJIT applauds students for studies on brain injury, glaucoma and more 2Hunger hormone: Makes food more attractive 2Finding the real potential of no-till farming for sequestering carbon 2
MAb to Hu-IFN-Gamma RC2 (CD118), NON-Neutralizing, Clone MMHGR-2 Binds to but does not neutralize human interferon gamma receptor...
Recombinant Rat CINC-2 beta, CF...
SHEEP ANTI FLECAINIDE...
RAT ANTI MOUSE F4/80 ANTIGEN:BIOTIN Immunogen: Thioglycollate stimulated peritoneal macrophages from C57/BL mice...
Biology Products: