Plasmid isolation

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.

1. Review on Isolation of Plasmid
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2. Contents Overview
Introduction to Plasmids
Literature Reviews
Plasmid Propagation
Importance of Plasmids
Electron Micrograph of DNA from lysed E.Coli cell.
Requirements for Plasmid Isolation.
Purification of plasmid DNA
Gel Electrophoresis
Conclusion
References
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3. Introduction to
Plasmids
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.
3

4. Literature Reviews
E. coli Plasmid Vectors: Methods in Molecular Biology.
Casali N, Preston A
Public Health England (PHE) National Mycobacterium Reference Laboratory, Clinical TB and HIV
Group, Blizard Institute, Queen Mary University of London, London, UK
(2010) Humana Press, Totowa, NJ, USA.
Manipulation of recombinant DNA, which is almost exclusively performed using the host E. coli,
constitutes one of the fundamental methodologies of molecular biotechnology. In E. coli Plasmid
Vectors, experienced bench researchers describe their proven techniques for the manipulation of
recombinant plasmids utilizing this popular bacterial host. The authors describe readily reproducible
methods for cloning DNA into plasmid vectors, transforming plasmids into E. coli, and analyzing
recombinant clones. They also include protocols for the construction and screening of libraries, as well
as specific techniques for specialized cloning vehicles, such as cosmids, bacterial artificial
chromosomes, l vectors, and phagemids. Also presented are methods for common downstream
applications, such as mutagenesis, expression of recombinant proteins and RNA transcripts, and uses
of reporter genes. Each fully tested protocol is described in systematic detail by an established expert in
the field and includes an introduction outlining the principles behind the technique, lists of the necessary
equipment and reagents, tips on troubleshooting and avoiding known pitfalls and, where needed, a
discussion of the interpretation and use of the results. Comprehensive and highly practical, E. coli
Plasmid Vectors offers those new to the field a basic guide to the use of plasmid vectors in the cloning
host E. coli, and those more experienced researchers a broad-ranging, proven array of successful
techniques
[1]
.
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5. Nature of Col E1 Plasmid Replication in Escherichia coli in the Presence of
Chloramphenicol.
Don B. Clewell
Departments of Oral Biology and Microbiology, the University of Michigan, Ann Arbor,
Michigan
(1994) Journal of Bacteriology.
The colicinogenic factor E1 (Col E1) in Escherichia coli continues to replicate by a semiconservative
mechanism in the presence of chloramphenicol (CAP) for 10 to 15 hr., long after chromosomal
deoxyribonucleic acid (DNA) synthesis has terminated. Following CAP addition, the rate of synthesis
of plasmid DNA gradually increases to an extent dependent on the medium employed. Within 2 to 4
hr after the addition of CAP, replication in a glucose-Casamino Acids medium approaches a
maximum rate representing approximately eight times an average rate which would be required for a
net doubling of DNA per cell in one generation. The number of copies of Col E1 DNA molecules that
accumulate under these conditions approaches about 3,000 copies per cell, representing a 125-fold
increase over the normal level of 24 copies per cell. The system is particularly convenient for
studying the mechanism of DNA replication
[2].
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Literature Reviews

6. Plasmid Propagation
•The plasmid DNA can replicate in bacteria and therefore many copies of the
plasmid will be made. The human DNA fragment in the plasmid will also
multiply along with the plasmid DNA.
•Once ligated into a plasmid, unlimited copies of a single gene can be
produced. The process of amplifying and isolating the human DNA fragment
is called DNA cloning.
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Importance of Plasmids
Most genes are present as two copies in the entire genome. Plasmids allow us
to obtain 1000’s of copies of a gene in a pure form.

8. E. coli
Chromosome
Plasmids
Plasmids
• DNA molecules separate from chromosomal DNA
• Self-replicating
Electron micrograph of DNA
from a lysed E. coli cell
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Purification of plasmid DNA
Multiple methods of nucleic acid purification exist.
•Ethanol precipitation
Ethanol precipitation works by using ethanol as an ant solvent of DNA, causing it to
precipitate out of solution. The soluble fraction is discarded to remove other
biomolecules.
•Spin column
Spin column-based nucleic acid purification precipitates nucleic acid such that it binds
a solid matrix and other components flow through. The conditions are then changed to
elute the purified nucleic acid.
•Phenol–chloroform extraction
In a phenol–chloroform extraction, addition of a phenol/chloroform mixture will
dissolve protein and lipid contaminants, leaving the nucleic acids in the aqueous
phase. It also denatures proteins, like DNase, which is especially important if the
plasmids are to be used for enzyme digestion. Otherwise, smearing may occur in
enzyme-restricted form of plasmid DNA.

•Analysis of plasmid DNA by gel electrophoresisAnalysis of plasmid DNA by gel electrophoresis

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Gel
Electrophoresis
Figure : Gel electrophoresis apparatus.

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In virtually all bacterial species, plasmids exist. These accessory genetic
elements typically account for only a small fraction of a bacterial genome
corresponding roughly to a range between 1 and 200 kb. Extremely large
plasmids with sizes far beyond 200 kb are also known. Plasmids of more
than 50 kb might be characterized as “large”, plasmids of less than 10 kb as
“small”. The aim of this compilation is to describe some fast methods for
small-scale plasmid isolation leading to “crude lysates”, the quality of which
being sufficient for analytical purposes, mainly agarose gel electrophoresis.
Further downstream steps might require a higher quality of plasmid DNA
and therefore, additional purification.

A plasmid  preparation is  a  method  of DNA  extraction and  purification 
for plasmid  DNA.  Many  methods  have  been  developed  to  purify 
plasmid DNA from bacteria.  These  methods  invariably  involve  three  steps: 
Growth of the bacterial culture, Harvesting and lysis of the bacteria,
Purification of plasmid DNA.
Conclusion

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1. Casali N, Preston A, E. coli Plasmid Vectors. Methods in Molecular
Biology, Humana Press, Totowa, NJ, USA; 235, 2010: 23-39.
2. Clewell DB, Nature of ColE1 plasmid replication in Escherichia coli
in the presence of chloramphenicol, Journal of Bacteriology; 110,
1994: 667-676.
3. Crosa JH, Tolmasky ME, Actis LA, Falkow, Plasmids- Methods for
general and molecular bacteriology, American Society for
Microbiology, Washington 1994: 365-386.
4. Davis RW, Botstein D, Roth JR, Advanced bacterial genetics. A
manualfor genetic engineering, Cold Spring Harbor Laboratory Press;
Cold Spring Harbor, 1980: NY, USA.
5. Eckhardt T, A rapid method for the identification of plasmid
deoxyribonucleic acid in bacteria, Plasmid; 1(2), 1971: 584-588.
6. Funnell BE, Phillips GJ, Plasmid Biology, ASM Press; Washington,
DC, USA, 2004.
References

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