It typically exists as a covalently closed circular piece of double stranded DNA that has the capability of replicating autonomously and it is this property that leads to its isolation and physical recognition. The closed covalent nature of their structure allows them to be separated from chromosomal DNA by either gel electrophoresis or cesium chloride buoyant density gradients.
Plasmid is a double stranded, circular extra chromosomal DNA of bacterium. It is used in recombinant DNA experiments to clone genes from other organisms and make large quantities of their DNA. Plasmid can be transferred between same species or between different species. Size of plasmids range from 1-1000 kilo base pairs. Plasmids are part of mobilomes (total of all mobile genetic elements in a genome) like transposons or prophages and are associated with conjugation. Even the largest plasmids are considerably smaller than the chromosomal DNA of the bacterium, which can contain several million base pairs.
Schematic drawing of a bacterium with its plasmids.
(1) Chromosomal DNA . (2) Plasmids

Plasmid DNA may appear in one of the five conformations, which run at different speeds in a gel during electrophoresis. The different plasmid conformations are listed below in the order of electrophoretic mobility .
Nicked Open-Circular DNA ,which has one strand cut.
Relaxed Circular DNA is fully intact with both strands uncut, but has been enzymatically relaxed.

Linear DNA has free ends, either because both strands have been cut, or because the DNA was linear in vivo.
Super coiled (or Covalently Closed-Circular) DNA is fully intact with both strands uncut, and with a twist built in, resulting in a compact form.

Super coiled Denatured DNA is like super coiled DNA, but has unpaired regions that make it slightly less compact; this can result from excessive alkalinity during plasmid preparation.

The relative electrophoretic mobility (speed) of these DNA conformations in a gel is as follows:
Nicked Open Circular ( slowest )
Linear
Relaxed Circular
Supercoiled Denatured
Supercoiled ( fastest )

Types of Bacterial Plasmids
Based on their function, there are five main classes:
Fertility-(F)plasmids: they are capable of conjugation or mating.
Resistance-(R) plasmids: containing antibiotic or drug resistant gene(s). Also known as Rfactors, before the nature of plasmids was understood.
Col-plasmids: contain genes that code for colicines , proteins that can kill other bacteria.
Degrative plasmids: enable digestion of unusual substances, e.g., toluene or salicylic acid .
Virulence plasmids: turn the bacterium into a pathogen .

Amp-R
Antibiotic resistance ori
Kan-R
Schematic drawing of a plasmid with antibiotic resistances
R-plasmids often contain genes that confer a selective advantage to the bacterium hosts, e.g., the ability to make the bacterium antibiotic resistant .
Some common antibiotic genes in plasmids: amp r , APH3’-II (kanamycin), tet R
(tetracycline),cat R (Chloramphenicol), spec r (spectinomycin or streptomycin), hyg r
(hygromycin) .
Some antibiotics inhibit cell wall synthesis and others bind to ribosomes to inhibit protein synthesis

Application of Plasmid Vectors
In Pharmaceutical and Agriculture Bioengineering
One of the major uses of plasmids is to make large amounts of proteins.
In this case, bacteria or other types of host cells can be induced to produce large amounts of proteins from the plasmid with inserted gene, just as the bacteria produces proteins to confer antibiotic resistance. This is a cheap and easy way of mass-producing a gene or the protein — for example,
.

How to rapidly isolate plasmid?
(a) Inoculation and harvesting the bacteria
(b) lysis of the bacteria (heat, detergents (SDS or
Triton-114), alkaline(NaOH)),
(c) neutralization of cell lysate and separation of cell debris (by centrifugation),
Or other cell types

Tranditional Ways
continued
Midi Prep Mini Prep
(d) collecting plasmid DNA by centrifugation ( after ethanol precipitation or through filters - positively charged silicon beads),
(e) check plasmid DNA yield and quality (using spectrophotometer and gel electrophoresis ).
spectrophotometer and gel electrophoresis

Procedure
• Fill a microcentrifuge tube with saturated bacterial culture grown in LB broth + antibiotic. Spin tube in microcentrifuge for 1 minute, and make sure tubes are balanced in microcentrifuge.
Dump supernatant and drain tube briefly on paper towel.
Repeat step 1 in the same tube, filling the tube again with more bacterial culture. The purpose of this step is to increase the starting volume of cells so that more plasmid DNA can be isolated per prep. Spin tube in microcentrifuge for 1 minute. Pour off supernatant and drain tube on paper towel.
Add 0.2 ml ice-cold Solution 1 to cell pellet and resuspend cells as much as possible using disposable transfer pipet.
Solution 1 contains glucose, Tris, and EDTA. Glucose is added to increase the osmotic pressure outside the cells. Tris is a buffering agent used to maintain a constant pH ( = 8.0).
EDTA protects the DNA from degradative enzymes (called DNAses); EDTA binds divalent cations that are necessary for DNAse activity.
Add 0.4 ml Solution 2, cap tubes and invert five times gently. Let tubes sit at room temperature for 5 minutes.
Solution 2 contains NaOH and SDS (a detergent). The alkaline mixtures ruptures the cells, and the detergent breaks apart the lipid membrane and solubilizes cellular proteins. NaOH also denatures the DNA into single strands.

Add 0.3 ml ice-cold Solution 3, cap tubes and invert five times gently. Incubate tubes on ice for 10 minutes.
Solution 3 contains a mixture of acetic acid and potassium acetate. The acetic acid neutralizes the pH, allowing the DNA strands to renature. The potassium acetate also precipitates the SDS from solution, along with the cellular debris. The E.
colichromosomal DNA, a partially renatured tangle at this step, is also trapped in the precipitate. The plasmid DNA remains in solution.
Centrifuge tubes for 5 minutes. Transfer supernatant to fresh microcentrifuge tube using clean disposable transfer pipet. Try to avoid taking any white precipitate during the transfer.
It is okay to leave a little supernatant behind to avoid accidentally taking the precipitate.
This fractionation step separates the plasmid DNA from the cellular debris and chromosomal DNA in the pellet.
Fill remainder of centrifuge tube with isopropanol. Let tube sit at room temperature for 2 minutes.

Isopropanol effectively precipitates nucleic acids, but is much less effective with proteins. A quick precipitation can therefore purify DNA from protein contaminants.
.
Centrifuge tubes for 5 minutes. A milky pellet should be at the bottom of the tube. Pour off supernatant without dumping out the pellet. Drain tube on paper towel.
This fractionation step further purifies the plasmid DNA from contaminants. This is also a good place to stop if class time is running out. Cap tubes and store in freezer until next class period.
Add 1 ml of ice-cold 70% ethanol. Cap tube and mix by inverting several times. Spin tubes for 1 minute. Pour off supernatant (be careful not to dump out pellet) and drain tube on paper towel.
Ethanol helps to remove the remaining salts and SDS from the preparation.
Allow tube to dry for ~5 minutes. Add 50 ul TE to tube. If needed, centrifuge tube briefly to pool
TE at bottom of tube. DNA is ready for use and can be stored indefinitely in the freezer

.
Solution 1:
50 mM glucose
25 mM Tris-HCl pH 8.0
10 mM EDTA pH 8.0
Add H
2
O to 500 ml.
Solution 2:
1% SDS
0.2 N NaOH
Solution 3:
3 M K+
5 M Acetate per 500 ml:
9 ml 50% glucose
12.5 ml 1 M Tris-HCl pH 8.0
10 ml 0.5 M EDTA pH 8.0
per 500 ml:
50 ml 10% SDS
100 ml 1 N NaOH per 500 ml:
300 ml 5 M Potassium Acetate
57.5 ml glacial acetic acid
Add H
2
O to 500 ml.

TE
10 mM Tris-HCl pH 8.0
1 mM EDTA
Add H
2
O to 100 ml per 100 ml:
1 ml 1 M Tris-HCl pH 8.0
0.5 ml 0.5 M EDTA pH 8.0
.Optional: RNAse can be added to TE at final concentration of 20 ug/ml.