Isolation of Plasmid DNA
Introduction
Many variations on a theme exist for the isolation of plasmid DNA from bacterial cells. They
usually involve the following steps (not necessarily in this order):
a. growing cells under conditions allowing intracellular accumulation of large amounts of
plasmid; harvesting the cells by centrifugation;
b. cell lysis (often using lysozyme to cleave cell wall peptidoglycans and surfactants to
dissolve cell walls/membranes);
c. separation of chromosomal DNA; removal of cellular RNA with ribonuclease;
d. deproteinization;
e. precipitation of the plasmid DNA with alcohol.
EDTA is included in most solutions to chelate Mg2+ ions and thus inhibit deoxyribonucleases
that would otherwise degrade the plasmid DNA (RNases do not require Mg2+ as a rule).
We will use a recently-reported method designed for medium scale isolation that is quick and
that yields good quality plasmid DNA, free of RNA, chromosomal DNA and impurities that
interfere with restriction enzymes and other subcloning operations.
Ausebel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K.
(eds.) 1989. In: Short Protocols in Molecular Biology, pp. 22-23, John Wiley & Sons, New York.
We will prepare plasmid pO.380, a vector which contains 3.8 kb of Drosophila DNA subcloned
into the EcoRI restriction site within the polylinker region of pBluescribe (Stratagene) (McLean,
J.R., Boswell, R., and O’Donnell, J., 1990. Cloning and molecular characterization of a
metabolic gene with developmental functions in Drosophila: Analysis of the head function of
Punch. Genetics 126:1007 - 1019). The strain that harbors this plasmid is ampicillin-resistant and
therefore may be selectively grown in medium containing this antibiotic. It is in general
advisable to grow plasmid-containing strains under selective conditions (e.g., in the presence of
an appropriate antibiotic) to insure that the plasmids are not eliminated from the cells.
Background Notes
Bacterial plasmids are double-stranded closed circular (supercoiled) DNA molecules that range
in size from 1 kb to 200 kb.
Plasmids are found in a variety of bacterial species.
Plasmids behave as accessory genetic units that replicate and are inherited independently of the
bacterial chromosome (extrachromosomal genetic elements).
Plasmids rely on cellular enzymes and structural proteins for their replication and transcription.
Plasmids can confer a selective advantage to the host cell (resistance to antibiotics).
History: Dr. Lederberg in 1952 first discovered plasmids (F factor) (sex factor) in bacteria.
F factor is a special type of plasmid = episome
Episome is a segment of DNA capable of existing in 2 alternate forms, one replicating
autonomously in the cytoplasm (plasmid), the other replicating as part of the bacterial
chromosome (DNA can integrate into the host genome).
Current plasmid vectors are derived from a plasmid isolated from a clinical specimen in the
1970's (pMB1).
pBluescribe (also pUC) is a derivative of pMB1.
Mobilization and Selectable Markers of Plasmids:
Under natural conditions, plasmids are transmitted to a new host by a process called bacterial
conjugation.
In the laboratory, plasmid DNA can be introduced into bacteria by the artificial process of
transformation.
In this process, bacteria are treated with divalent cations (Ca2+) to make them temporarily
permeable to small DNA molecules.
Efficiency of transformation is very low, so in order to identify transformed cells (cells which
incorporated plasmids via transformation), one monitors a selectable marker which is encoded by
the plasmid.
A selectable marker confers a new phenotype to the host cell that allows bacteria that have been
successfully transformed to be selected (with ease).
Commonly used selectable markers are genes that confer resistance to antibiotics.
Ampicillin: inhibits enzymes that are involved in the synthesis of the cell wall.
Tetracycline: binds to a protein on the 30S subunit of the ribosome and inhibits ribosomal
translocation.
Chloramphenical: binds to the 50S subunit of the ribosome and inhibits protein synthesis.
Kanamycin and neomycin: are deoxystreptamine aminoglycosides that bind to ribosomal
components and inhibit protein synthesis.
pBluescribe (pUC) carries an ampicillin resistance gene. (bla) = encodes an enzyme that is
secreted into the cell wall and breaks down ampicillin
Today's experiment (and next week's) will follow a procedure to isolate a pBluescribe plasmid.
pBluescribe has a polycloning site (a DNA sequence that was genetically constructed in vitro
and contains many sites which are recognized by restriction endonucleases (enzymes): one of a
large number of nucleases (enzymes that degrade nucleic acids) that can cleave a DNA molecule
at any site where a specific short sequence of nucleotides occurs. Restriction enzymes are
extensively used in recombinant DNA technology.
pBluescribe also contains a selectable marker (bla) so it confers an ampicillin resistant
phenotype.
General Procedure:
Cells containing plasmids are concentrated and resuspended in an isotonic solution of sucrose.
Cells containing plasmids are treated with lysozyme to break down the cell wall and outer
membrane.
Plasmids are released from cells by gentle lysis (addition of a nonionic detergent).
Cell lysate is heated to release carbohydrates and nick chromosomal DNA.
Plasmid DNA, RNA and proteins are concentrated by alcohol precipitation.
Proteins are separated from plasmid DNA and RNA with saturated phenol.
Phenol removed with chloroform.
Plasmid DNA and RNA are concentrated by alcohol precipitation.
RNA removed with RNase.