lab2-plasmid isolation (1)
TRANSCRIPT
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Microbial Genetics (GEN 203)
Lab Two
Plasmid Isolation
Course Coordinator: Dr. Ashraf Bakkar
L.As: Ingy El-Hefny, Heba Hisham , T.A: Aya Farghally
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What are plasmids?
• A plasmid is distinct or independent from a cell’s
chromosomal DNA.It’s main characteristics are:
small and circular
double-stranded DNA molecule
Self replicating
• Plasmids naturally exist in bacterial cells only, right?
• WRONG! They can occur in some eukaryotes too such as
Entamoeba histolytica, yeast etc.
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1) The bacterial chromosome: DNA of most bacteria is contained in
a single circlar molecle, called the bacterial chromosome.2) The nucleoid: is an irreglar sha!e that contains se"eral !roteins
and #NA molecles
3) Plasmids: small circlar DNA molecles.
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Types of Plasmids
• Plasmids are not usually required by their host cell for its survival.
Instead, they carry genes that confer aselective advantage on their host.
• Plasmids are classified into five main types according to that
ADVANTAGE:
1.Fertility F-plasmids:which containstra genes. They provide the
fertility system required for conjugation. Sometimes they are called
episomes.
2.Resistance (R)plasmids:R plasmids carry genes encoding resistance
to naturally made antibiotics by other microorganisms or to toxic
substanaces such as heavy metals
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3. Col plasmids:Col plasmids allows their host to produce
antibacterial polypeptides called bacteriocins that are often
lethal to closely related or other bacteria. The col proteins ofE.
coli are encoded by plasmids such as ColE1.
4. Degradative plasmids: allow a host cell to metabolize
normally undegradable or difficult compounds such as
various pesticides.
5. Virulence plasmids:which turn the bacterium into a pathogen
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How can we isolate plasmids?
• First:we isolate plasmids from the bacterial cells bythe
alkaline lysis methodwhich depends on a unique property
of plasmid DNA; it’s ability to rapidly anneal following
denaturation. This is what allows the plasmid DNA to be
separated from the bacterial chromosome.
• Second: we purify it from the rest of the cellular components
bythe silica based membrane technology
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Alkaline Lysis
1. Harvest bacterial Cells: Bacterial cells are pelleted by centrifugation to
remove them from the growth medium.
2. Re-suspension: The pellet is then re-suspended in a solution containing
often Tris, EDTA, and RNase A.
Function of:
A)EDTA:chelates divalent cations such as (Mg2+, Ca2+) in the solution
preventing DNases from damaging the plasmid and also helps by
destabilizing the cell wall.
B) RNase A:to degrade cellular RNA when the cells are lysed.
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3. Lysis solution:contains sodium hydroxide (NaOH) and the detergent Sodium
Dodecyl (lauryl) Sulfate (SDS).
Function of:
A)SDS:is there to solubilize the cell membrane and denatures proteins.
B) NaOH:helps to break down the cell wall, but more importantly it denatures
DNA by disrupting the hydrogen bonding between the DNA bases, converting
the double-stranded DNA (dsDNA) in the cell, including the genomic DNA
(gDNA) and your plasmid, to single stranded DNA (ssDNA). This is why it’s
calledalkaline lysis.
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4. Neutralization solution:contains potassium acetate
Potassium acetate has two functions:
1)Re-naturation of plasmids:it decreases the alkalinity of the mixture, so
the hydrogen bonding between the bases of the single stranded DNA can
be re-established. It is only easy for the small circular plasmid DNA to
re-nature.
2)Precipitates unwanted cell debris and gDNA:large ssDNA are insoluble
in high salts, while the double-stranded plasmid can dissolve easily in
solution. As a result the single stranded genomic DNA, the SDS and the
denatured cellular proteins will form a white precipitate.
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Now your plasmid DNA has been separated from themajority of the cell debris but is in a solution
containing lots of salt, EDTA, RNase and residual
cellular proteins and debris, so it’s not much use
for downstream applications.
The next step is to clean up the solution and
concentrate the plasmid DNA.
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Silica Membrane Based Technology
• Principle:
• Chaotrophic salts are added to the sample to denature the DNA by disrupting it’s
hydrogen bonding.
• Under these conditions, the DNA will selectively bind to the silica resin because
hydrogen bonds will form between the membrane and the phosphate backbone.
• After washing the DNA is eluted
from the column with a low salt
solution which allows the re-
naturing of the DNA, causing it to
lose affinity for the silica.
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Smmary
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!ow yor plasmid is ready for downstream
applications
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NEXT WEEK
• Possible troubleshoots
• Detect your plasmid!
• Different Plasmids Structure and plasmid
Replication• Difference between Plasmids and Vectors
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THA!" #$%