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Plasmid Isolation
what is a plasmid ?
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.
Conformation of Plasmid DNAs
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: ampr, APH3’-II (kanamycin), tetR
(tetracycline),catR (Chloramphenicol), specr (spectinomycin or streptomycin), hygr
(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, insulin, antibiotics,
antobodies and vaccines.
Plasmid Isolation from Bacteria
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
Plasmid DNA Isolation continued
Tranditional Ways
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
.
.
Solutions:
Solution 1:
per 500 ml:
50 mM glucose
9 ml 50% glucose
25 mM Tris-HCl pH 8.0
12.5 ml 1 M Tris-HCl pH 8.0
10 mM EDTA pH 8.0
10 ml 0.5 M EDTA pH 8.0
Add H2O to 500 ml.
Solution 2:
per 500 ml:
1% SDS
50 ml 10% SDS
0.2 N NaOH
100 ml 1 N NaOH
Solution 3:
per 500 ml:
3 M K+
300 ml 5 M Potassium Acetate
5 M Acetate
57.5 ml glacial acetic acid
Add H2O to 500 ml.
TE
per 100 ml:
10 mM Tris-HCl pH 8.0
1 ml 1 M Tris-HCl pH 8.0
1 mM EDTA
0.5 ml 0.5 M EDTA pH 8.0
Add H2O to 100 ml
.Optional: RNAse can be added to TE at final concentration of 20
ug/ml.
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