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Plasmid DNA Isolation
Experiment Goals
• Extraction of plasmid DNA from E. Coli.
• Analyze plasmid DNA by agarose gel
electrophoresis and spectrophotometer.
Introduction
• Many types of bacteria contain plasmid DNA.
• Plasmids
are extrachromosomal, double-stranded
circular DNA molecules separate from the
chromosomal DNA.
• Certain plasmids replicate independently of the
chromosomal DNA and can be present in hundreds of
copies per cell.
• Generally containing1,000 to 100,000 base pairs.
• Even the largest plasmids are considerably smaller
than the chromosomal DNA of the bacterium, which
can contain several million base pairs.
Classification of plasmids by function
There are five main classes
• Fertility-F-plasmids, Facilitate bacterial conjugation
• Resistance-R-plasmids, which contain genes that can
build a resistance against antibiotics or poisons.
• Col-plasmids, which contain genes that code for
bacteriocins, proteins that can kill other bacteria.
• Degradative plasmids, which enable the digestion of
unusual substances, e.g., salicylic acid.
• Virulence plasmids, which turn the bacterium into a
pathogen.
Plasmid Applications
• The plasmids used in transformation typically
have three important elements:
• A cloning site (a place to insert foreign
DNAs)
• An origin of replication
• A selectable marker gene (e.g. resistance to
ampicillin)
Plasmid Applications
Plasmid DNA isolation
• Isolation of plasmid DNA from bacterial cells is an
essential step for many molecular biology procedures.
• Many protocols for large- and small-scale isolation of
plasmids have been published.
• The plasmid purification procedures, unlike the
procedures for purification of genomic DNA, should
involve removal not only of protein, but also another
major impurity: bacterial chromosomal DNA.
Overnight Culture Suspension
• Pick a single colony and inoculate
in 5 ml of LB (Luria-Bertani)
containing 20 mg/l ampicilin
• Incubate overnight at 37oC
• Centrifuge 1.5 ml of
containing cells in a tube
• Discard supernatant
broth
Plasmid DNA isolation
1. Inactivation of Bacteria
2. Lysis of cells/ denaturation of DNA
3. Precipitation of DNA
4. Separate plasmid DNA from contaminants
5. Precipitation of Plasmid DNA
6. Precipitation of proteins
7. Precipitate Plasmid DNA
1- Inactivation of Bacteria
• Resuspend cell pellet in 100 µl of GTE buffer
(50mM Glucose, 25 mM Tris-Cl & 10mM EDTA,
pH 8)
• Glucose is added to increase the osmotic pressure
outside the cells
• Tris is a buffering agent
• EDTA protects the DNA from degradative
enzymes
• Vortex gently if necessary
2- Lysis of cells/ denaturation of DNA
Add 200 µl of NaOH/ SDS lysis solution, invert tube 6-8 times
1. Sodium dodecyl sulfate
• Dissolves membranes
• Binds to and denatures proteins
2. NaOH
• NaOH rupture the cell and also denatures the DNA into single strands
3- Precipitation of DNA
• Immediately add 150 µl of 5 M potassium acetate solution (pH 4.8)
1. Potassium acetate / acetic acid solution
• Neutralizes NaOH (renature plasmid DNA)
• Converts soluble SDS to insoluble PDS
sodium dodecyl sulfate (SDS)
• Precipitate the genomic DNA
• Centrifuge for 1 minute at high speed
potassium dodecyl sulfate (PDS)
4- Separate plasmid DNA from contaminants
Separate plasmid DNA from contaminants by
centrifugation
•
Supernatant contains:
- Plasmid DNA
- Some cellular constituents
•
Sediment contains:
- PDS
- Lipids
- Proteins
- Chromosomal DNA
5- Precipitation of Plasmid DNA
• Transfer supernatant layer to a clean tube and add 0.5 ml of
isopropanol on ice for 10 minutes
• Centrifuge at top speed for 1 minute
Add 0.5 ml of
isopropanol to
supernatant
Incubate for 10
min. on ice
Supernatant
Centrifuge
Pellet
• Remove supernatant, dissolve pellet in 0.4 ml TE buffer
• Add 10 µl of RNAse solution, vortex & incubate at 37oC for 20
– 30 min.
6- Precipitation of proteins
• Add 300 µl of phenol/ chloroform/ Isoamyl alcohol
• Vortex vigorously for 30 seconds
• Centrifuge at full speed for 5 minutes
Mix thoroughly with
an equal volume of
organic solvent
Aqueous
Centrifuge
phenol, chloroform,
Organic
7- Precipitate Plasmid DNA
• Remove supernatant to a clean tube
• Add 100 µl of 7.5 M ammonium acetate & 1 ml of absolute
ethanol to precipitate the plasmid DNA, incubate on ice
• Mix and then centrifuge at full speed for 5 minutes
Supernatant
Absolute ethanol & ammonium acetate
Centrifuge
precipitated DNA
Pellet
• Wash pellet with 75% Ethanol (to remove salts), & dry pellet
• Dissolve pellet with TE (or other aqueous solution)
Quantifying Plasmid DNA
• Quantify DNA using UV absorbance
• DNA UV absorbance peaks at 260 nm
• protein UV absorbance peaks at 280 nm
• The ratio of the absorbance at 260 nm/280 nm is a measure of
the purity of a DNA sample from protein contamination; it
should be between 1.7 and 2.0
• The ratio of the absorbance at 260 nm/230 nm is a measure of
the purity of a DNA sample from organics and/or salts; it
should be about 2.0. Low 260/230 ratio indicates
contamination by organics and/or salts
• http://www.dnatube.com/video/994/Plasmid•
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Isolation
http://www.dnalc.org/resources/animations/transf
ormation1.html
http://www.learnerstv.com/animation/animation.p
hp?ani=167&cat=biology
http://resources.jorum.ac.uk/xmlui/bitstream/hand
le/123456789/13704/page83.htm?sequence=86
http://www.sumanasinc.com/webcontent/animatio
ns/content/dna_library.swf
http://www.sumanasinc.com/webcontent/animatio
ns/content/plasmidcloning.html