ISOLATION OF PLASMID DNA FROM E.Coli


The plasmid:
A plasmid is a small circular piece of DNA (about 2,000 to 10,000 base pairs) that contains important genetic information for the growth of bacteria. In nature, this information is often a gene that encodes a protein that will make the bacteria resistant to an antibiotic.

Plasmids were discovered in the late sixties, and it was quickly realized that they could be used to amplify a gene of interest. A plasmid containing resistance to an antibiotic (usually ampicillin) is used as a vector. The gene of interest is inserted into the vector plasmid and this newly constructed plasmid is then put into E. coli that are sensitive to ampicillin. The bacteria are then spread over a plate that contains ampicillin. The ampicillin provides a selective pressure because only bacteria that have acquired the plasmid can grow on the plate. Therefore, as long as you grow the bacteria in ampicillin, it will need the plasmid to survive and it will continually replicate it, along with your gene of interest that has been inserted to the plasmid.

There are many different kinds of plasmids commercially available. All of them contain
1) A selectable marker (i.e., a gene that encodes for antibiotic resistance),
2) An origin of replication (which is used by the DNA making machinery in the bacteria as the starting point to make a copy of the plasmid) and
3) A multiple cloning site. The multiple cloning site has many restriction enzyme sites (to be discussed in a later lab) and is used to insert the DNA of interest.

Conformations
Plasmid DNA may appear in one of five conformations, which (for a given size) run at different speeds in a gel during electrophoresis. The conformations are listed below in order of electrophoretic mobility (speed for a given applied voltage) from slowest to fastest:
• "Nicked Open-Circular" DNA has one strand cut.
• "Relaxed Circular" DNA is fully intact with both strands uncut, but has been enzymatically "relaxed" (supercoils removed). You can model this by letting a twisted extension cord unwind and relax and then plugging it into itself.
• "Linear" DNA has free ends, either because both strands have been cut, or because the DNA was linear in vivo. You can model this with an electrical extension cord that is not plugged into itself.
• "Supercoiled" (or "Covalently Closed-Circular") DNA is fully intact with both strands uncut, and with a twist built in, resulting in a compact form. You can model this by twisting an extension cord and then plugging it into itself.
• "Supercoiled Denatured" DNA is like supercoiled DNA, but has unpaired regions that make it slightly less compact; this can result from excessive alkalinity during plasmid preparation. You can model this by twisting a badly frayed extension cord and then plugging it into itself.

The rate of migration for small linear fragments is directly proportional to the voltage applied at low voltages. At higher voltages, larger fragments migrate at continually increasing yet different rates. Therefore the resolution of a gel decreases with increased voltage. At a specified, low voltage, the migration rate of small linear DNA fragments is a function of their length. Large linear fragments (over 20kb or so) migrate at a certain fixed rate regardless of length. This is because the molecules 'reptate', with the bulk of the molecule following the leading end through the gel matrix. Restriction digests are frequently used to analyse purified plasmids. These enzymes specifically break the DNA at certain short sequences. The resulting linear fragments form 'bands' after gel electrophoresis. It is possible to purify certain fragments by cutting the bands out of the gel and dissolving the gel to release the DNA fragments. Because of its tight conformation, supercoiled DNA migrates faster through a gel than linear or open-circular DNA.

A plasmid is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. In many cases, it is circular and double-stranded. Plasmids usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms (e.g., the 2-micrometre-ring in Saccharomyces cerevisiae) .Plasmid size varies from 1 to over 200 kilo base pairs (kbp). The number of identical plasmids within a single cell can be zero, one, or even thousands under some circumstances. Plasmids can be considered to be part of the mobilome, since they are often associated with conjugation, a mechanism of horizontal gene transfer.
The term plasmid was first introduced by the American molecular biologist Joshua Lederberg in 1952.Plasmids can be considered to be independent life-forms similar to viruses, since both are capable of autonomous replication in suitable (host) environments. However the plasmid-host relationship tends to be more symbiotic than parasitic (although this can also occur for viruses, for example with Endo viruses) since plasmids can endow their hosts with useful packages of DNA to assist mutual survival in times of severe stress. For example, plasmids can convey antibiotic resistance to host bacteria, who may then survive along with their life-saving guests who are carried along into future host generations.

Plasmids that exist only as one or a few copies in each bacterium are, upon cell division, in danger of being lost in one of the segregating bacteria. Such single-copy plasmids have systems which attempt to actively distribute a copy to both daughter cells. Some plasmids include an addiction system or "postsegregational killing system (PSK)", such as the hok/sok (host killing/suppressor of killing) system of plasmid R1 in Escherichia coli. They produce both a long-lived poison and a short-lived antidote. Daughter cells that retain a copy of the plasmid survive, while a daughter cell that fails to inherit the plasmid dies or suffers a reduced growth-rate because of the lingering poison from the parent cell.

Alkaline lysis was first described by Birnboim and Doly in 1979 (Nucleic Acids Res. 7, 1513-1523) and has, with a few modifications, been the preferred method for plasmid DNA extraction from bacteria ever since.

Solution 1 contains Tris, EDTA, glucose and RNase A. Divalent cations (Mg2+, Ca2+) are essential for DNase activity and the integrity of the bacterial cell wall. EDTA chelates divalent cations in the solution preventing DNases from damaging the plasmid and also helps by destabilizing the cell wall. Glucose maintains the osmotic pressure so the cells don't burst and RNase A is included to degrade cellular RNA when the cells are lysed.

The lysis buffer (solution 2) contains sodium hydroxide (NaOH) and the detergent Sodium Dodecyl (lauryl) Sulfate (SDS). SDS is there to solubilize the cell membrane. NaOH helps to break down the cell wall, but more importantly it disrupts the hydrogen bonding between the DNA bases, converting the double-stranded DNA (dsDNA) in the cell, including the genomic DNA and plasmid, to single stranded DNA (ssDNA). This process is called denaturation and is central part of the procedure ( alkaline lysis). SDS also denatures most of the proteins in the cells, which helps with the separation of the proteins from the plasmid later in the process.

Addition of potassium acetate (solution 3) returns the pH to neutral. Under these conditions the hydrogen bonding between the bases of the single stranded DNA can be re-established, so the ssDNA can re-nature to dsDNA.. The small circular plasmid DNA re-natures but it is impossible to properly anneal the huge genomic DNA stretches. This is why it's important to be gentle during the lysis step because vigorous mixing or vortexing will shear the gDNA producing shorter stretches that can re-anneal and contaminate your plasmid prep.

While the double-stranded plasmid can dissolve easily in solution, the single stranded genomic DNA, the SDS and the denatured cellular proteins stick together through hydrophobic interactions to form a white precipitate. The precipitate can easily be separated from the plasmid DNA solution by centrifugation.

EXPERIMENTAL PROCEDURE
AIM
To isolate the plasmid DNA from E.coli

PRINCIPLE
This isolation is based upon the release of high molecular weight molecules of DNA from disrupted cells cell walls and membranes dissociating nuclear protein complex by denaturation and proteolysis and separation of DNA from other macromolecules observed.

MATERIALS REQUIRED
Overnight culture of E.coli
Test tubes
Centrifuge
Ice pack
Eppendorf tubes

REAGENT PREPARATION

1. SOLUTION 1 - 5ml
50mM Tris HCl
Pipette out 0.25 ml of 1 M TrisHCl in a beaker
20mM EDTA
Pipette out 0.20 ml of 0.5 M EDTA into the beaker
15%Glucose
Weigh 0.75 grams of glucose
Dissolve the above chemicals and make it upto 5ml using distilled water

2. SOLUTION 2 - 5ml
0.2 N NaOH
Weigh 0.04 grams of NaOH in a beaker
1%SDS
Weigh 0.05 grams of SDS in a beaker
Dissolve the above solution in 3 ml of distilled water by heating and then make it upto 5 ml

3.SOLUTION 3 - 5ml
5M Pottasium acetate

PROCEDURE
• Take 1.5 ml of overnight culture of E.coli into Eppendorf tubes
• The tubes are then centrifuged at 6000rpm for 10 mins
• To this 100 microlitres of solution 1 was added and this was incubated in ice cold condition for 10 mins
• Then add 150 microlitres of solution 2 and gently vortex till a white viscous liquid is formed
• To this add 200 microlitres of solution 3 and incubate it in ice cold condition for 10 mins
• Then centrifuge the tubes at 10000 rpm for 10 min
• Then take supernatant and add equal volume of Isopropanol to that
• Incubate in ice cold condition for 30 min
• Centrifuge these tubes at 12000rpm for 10 mins
• The pellet was dried well and dissolve it in 50 microlitres of TE buffer
• Run the samples in a gel

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