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Isolation and purification of plasmids
#1
Being first coined by a molecular biologist in 1952 plasmids, the small, circular, extra chromosomal DNA mainly found in bacteria made its entry into the field of molecular biology as vectors in 1970s. Plasmids are able to self replicate without any dependence on the host chromosomal DNA, carry genes useful for the host organism (for example, antibiotic resistance gene) and also possess the ability to transfer among different bacterial species through horizontal gene transfer. These characteristic makes plasmids a useful vector tool in molecular biology in developing recombinant molecules. It is not that a single bacterial cell contains only one plasmid as there is a possibility of different plasmids existing in a single bacterial cell. Based on their ability to transfer between species plasmids are classified as conjugative and non-conjugative plasmids and another classification of plasmids made based on the function and the type of genes they carry include fertility plasmid, resistance plasmid, virulence plasmid, col plasmid and degradative plasmid.

With its wide application in molecular biology in the preparation of recombinants, plasmids need to be extracted or isolated from its host organism and thus isolated plasmids need to be purified. The initial step required in the extraction or isolation of the plasmid from the host bacteria is the preparation of the host bacterial culture which is called as minipreparation or miniprep from which the plasmid is extracted. As the name indicates miniprep involves preparation of few ml of the culture by inoculating the host organism containing the desired plasmid in the organism specific broth and allowing to grow overnight to attain the stationary phase. At this stage the culture is subjected to centrifugation, as a result the bacterial cells settle down forming a pellet which is then harvested and exposed to various processes like alkaline lysis, phenol extraction or cesium chloride gradient and ethanol precipitation to obtain pure plasmids.

In alkaline lysis, the harvested cell pellet is treated with buffer solution followed by the addition of special solution which initiates lysis of the cell and hence called as cell lysis solution. This solution is a combination of sodium dodecyl sulfate and sodium hydroxide, whereas sodium dodecyl sulfate acts on the cell membrane, lyses the membrane and also acts on the proteins by denaturing them and the alkaline environment created by the addition of the sodium hydroxide enables the denaturation and hydrolysis of the DNA and RNA respectively. The solution is then neutralized by the addition of potassium acetate which enables the precipitation of the proteins (denatured) and DNA (chromosomal) which upon centrifugation settles down leaving the plasmid DNA, RNA and fewer proteins in the supernatant.

The supernatant containing the plasmid, RNA and less protein is subjected to either phenol extraction method or cesium chloride gradient method to extract the plasmid alone from the mixed supernatant. The cesium chloride gradient is effective in obtaining pure plasmids and is adopted for plasmid production in large scale. In phenol extraction method, the addition of phenol to the supernatant followed by vigorous mixing denatures the proteins present in the supernatant which is finally seen as a separate precipitated layer below the plasmid - RNA layer. The plasmid - RNA layer is removed and processed with ethanol (ethanol precipitation) which enables the pelleting of the plasmid alone which is then treated with Tris hydrochloride and EDTA solution whereas the former is added to create buffer environment and the later takes care of any Magnesium ions present in the solution by chelating the ions and thus protects the plasmid DNA. The fear of RNA presence is cured by adding Ribonuclease A to the solution which takes care of the RNA and thus pure plasmid can be obtained.

In cesium chloride gradient method, as already said used for large scale plasmid production, the supernatant is treated with the solution of cesium chloride and ethidium bromide which upon centrifugation forms different fractions in the tube based on the density. The fractionation in the tube is seen as protein layer on top of all, followed by layer of chromosomal DNA, below which is the layer of super coiled plasmid and finally RNA is seen as pellets at the bottom of the tube. The plasmid layer is then recovered and subjected to ethanol precipitation method to obtain the pure plasmid.

Thus the plasmids with unique characteristics can be isolated and purified to be used as vectors in molecular biology.
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#2
Thanks for nice posting about the isolation and purification of Plasmids. Plasmid purification kits also bring great results with low levels of endotoxin being released for valued effects in the subject of transfer plasmids DNA.
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#3
A plasmid is an extra chromosomal DNA molecule quite different from the chromosomal DNA. Plasmid DNA is capable of replicating independently from the chromosomal DNA. They are double stranded and usually circular. Plasmids are known to be naturally occurring in bacteria and a few eukaryotic organisms.

Isolation of plasmid DNA is a common routine in research laboratories. The protocol involved in its extraction is called ‘mini prep’ which yields fairly clear DNA quickly and easily.Thie following protocol is based on the one given by Birnhoim and Doily in 1979 , it is modified according to the experiments’ requirements.

The varieties of chemical solutions used in the process of DNA extraction have different roles to play.

Solution I is added to maintain the pH of mixture containing the cells. SDS solution solubilises the phospholipids and proteins which are active components of cell membrane, thereby causing lysis of the cell and release of all its components. It contains RNase which acts on the RNA in the cell and denatures it.

Solution II is added for cell lysis in which NaOH is known to cause denaturation of chromosomal and plasmid DNA as well as the protein component of the cell.

Solution II is added for renaturation of plasmid DNA. For this purpose the mixture is made acidic by adding glacial acetic acid. The genomic DNA is very large so it can’t renature, hence only plasmid DNA is obtained.

Procedure:-
• Inoculate a test tube containing 100 ml of Luria Bertani media with a single isolated colony picked from LB agar plate.
• The culture is grown overnight at 37̊ C with shaking at regular intervals.
• 1.5 ml of overnight culture is centrifuged in a micro-centrifuge tube for 20 min at 4000 rpm.
• The supernatant is discarded and bacteria are resuspended in 5 ml of ice cold solution I by vortexing or pippetting up and down.
• Transfer the suspension to 50 ml ochrage tubes.
• 8 ml of solution II is added to it and mixed by shaking it up and down.
• Store at room temperature for 10 mins.
• 5 ml of solution III is added to it and thoroughly mixed. Store on ice for 30 mins.
• As soon as solution III is added, a white ppt is formed. The mixture is again centrifuged for 15 mins at 12000 rpm.
• The supernatant is then transferred to a fresh tube and 0.7 vol of isopropanol is then added to the supernatant and mixed properly.
• It is kept at room temperature for 10 mins and again centrifuged at 12000 rpm for 20 mins at 4̊ C.
• Discard the supernatant and air dry pellet.
• Add 1 ml in 100 ml of culture 1x TAE pH 8.0.
• Split into two eppendorfs. Add 250 µl phenol and 250 µl CHCl3.
• Mix by vortexing and centrifuge at 13000 rpm for 5 min.
• Take the aqueous phase and add 2 volume of chilled ethanol.
• Store at -20̊ C till required.
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#4
There are quick and easy method to isolate Plasmid from Bacteria here I have elaborated the protocol for isolation of Plasmid from different type of bacteria.

1. Boiling method for small plasmids in E. coli
This method is simple and developed by Holmes & Quigley in1981 and modified by Riggs & McLachlan, 1986.

• For the isolation of plasmid first centrifuge 1.5 ml of culture in micro centrifuge tube and re-suspend pellet in 200 μl of STET buffer (8.0% sucrose, 0.5% Triton X-100, 0.05 M EDTA, 0.05 M Tris-HCl, pH 8.0) containing 10 μl of lysozyme (20 mg/ml, freshly dissolved in H2O) and 20 μl ZnCl2 (1% in H2O).

• Incubate at about 100°C for 50-55 sec, and then cool on ice

• Centrifuge for 5-10 min and add supernatant to micro centrifuge tube containing 480 μl of IS mix (400 μl isopropanol, 80 μl 5 M ammonium acetate). Incubate at RT for 20-30 min.

• Centrifuge for 5 min, wash DNA pellet with 70% cold ethanol twice and dry in a vacuum chamber.

• Re-suspend pellet in 20 μl of storage TE buffer or in RNase buffer before using for DNA gel electrophoresis.

2. Hot alkaline method for all plasmid sizes and bacteria

This method was developed by Kado & Liu in1981

• For the plasmid isolation by this method, Centrifuge 2-3 ml of culture in micro centrifuge tube, re-suspend pellet in 1 ml of solution containing 0.04 M Tris-acetate, pH 8.0 (adjust pH with glacial acetic acid) and 2 mM EDTA.

• Add 2 ml of lysis buffer (0.05 M Tris, 3% SDS, pH 12.50, adjusted with 2 N NaOH) and mix well.

• Incubate at ~65°C for 30-45 min (strain dependent)

• Add to hot samples 6.0 ml of phenol/chloroform (1:1) and mix gently to complete emulsification.

• Separate phases by centrifugation at 10,000 RPM for 10-15 min at room temperature and transfer the upper aqueous phase carefully (avoid inter-phase which contains debris) to new tube containing 1.0 volume of chloroform. Mix and centrifuge again for separation of phases.

• Recover aqueous phase and use directly for DNA gel electrophoresis.

3. Lysozyme method for various Gram-negative bacteria

This method is simple and developed by Davis et al.,1980

• For the plasmid isolation by this method, centrifuge 10 ml of culture, re-suspend pellet in 1.4 ml of TE buffer (0.01 M Tris, pH 8.5 and 1 mM EDTA). Transfer to micro centrifuge tubes and spin for 3 min.

• Re-suspend pellet in 0.4 ml of solution (15% sucrose, 0.05 M Tris, pH 8.5, 0.05 M EDTA), mix vigorously and cool on ice.

• Add 0.1 ml of freshly prepared lysozyme (5 mg/ml in TE buffer used above), mix carefully and incubate on ice for 20-40 min.

• Add 0.3 ml of pre-cooled Triton buffer (0.1% Triton X-100, 0.05 M Tris, pH 8.5, 0.05 M EDTA), incubate on ice for 20 min and centrifuge at 4°C for 4 min.

• Transfer clear supernatant into new tube and add 4 μl of di-ethyloxydiformiate, mix gently.

• Incubate for 15 min at 70°C, cool for 15 min to RT, then incubate on ice for 15 min.

• Centrifuge it for 4 min, transfer supernatant into new tube, fill up with -20°C ethanol for DNA precipitation and mix gently.

• Centrifuge for at least 30 min at RT, dry the pellet in vacuum and re-suspend in storage TE buffer or in RNase buffer.


4. Lysis of cells from single colonies

This method was developed by Eckhardt, 1978 and Priefer, 1984

• For plasmid DNA isolation by this method, transfer 1-2 freshly grown single colonies with a toothpick into 20 μl of cold buffer (0.025 M Tris, pH 8.0, 25% sucrose, 0.250 M EDTA, 7% Ficoll 400).

• Add 20 μl of freshly prepared lysis solution (0.1 mg/ml of lysozyme, 10 μl/ml of RNase A, in the above buffer), mix well and immediately fill with 10-15 of the mixture into the well of an agarose gel which contains 0.5% SDS

• Add as “upper layer” onto the cell lysate 10 μl of the following solution: 0.025 M Tris, pH8.0, 10% SDS, 25% sucrose, 0.07% bromophenol blue

• After 15-30 min apply low voltage (half of usual voltage) for 30 min, then apply usual electrophoretical conditions

5. Plasmid isolation from Gram-positive bacteria with mutanolysin or lysozyme
This method was developed by Klaenhammer in 1984

• For the isolation of plasmid first centrifuge 4 ml of culture and re-suspend pellet in 10 ml of fresh culture medium and Incubate for 1-2 hrs at 37°C

• Centrifuge again and re-suspend pellet in 1 ml of cold 25% sucrose, 0.05 M Tris, pH 7.5, 5 mM EDTA at 4°C.

• Keep cell suspension in ice bath for 10 min, then add 75 μl of either mutanolysin or lysozyme (1 mg/ml in 0.05 M Tris, pH 7.5, 5 mM EDTA), mix and incubate in ice bath for 1hr (for some strains incubation at 37°C for 1hr is preferred)

• Centrifuge cells and add 500 μl of the following lysis solution to the pellet and mix well (0.05 M Tris, 5 mM EDTA, 0.05 M glucose, 3% SDS) immediately before use mix 1.0 ml of this solution with 10 μl of 10 N NaOH.

• Heat the sample at 62°C for 1 hr, then allow to cool slowly (approx.15 min) to RT, add 50 μl of 2 M Tris, pH 7.0, mix gently and add 70 μl of 5 M NaCl and mix gently.

• Transfer into micro centrifuge tube and extract with 500 μl of phenol which is saturated with 3% NaCl (mix gently until emulsification), leave at RT for 5-10 min. Add 300 μl of chloroform and mix gently.

• Centrifuge for 5 min at RT for phase separation and take upper phase for extraction with 600 μl of chloroform:isoamylalcohol which is (24:1), leave at RT for 5-10 min, centrifuge and harvest aqueous phase for ethanol precipitation as usual.
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Isolation and purification of plasmids00