Download Plasmid

Plasmid DNA isolation
Purpose :
 To learn how to extract plasmid DNA from E.coli.
 To observe the analysis of plasmid DNA by gel
electrophoresis.
Introduction :
 Over the past decades it became evident that virtually
in all bacterial species plasmids exist.
 These accessory genetic elements are defined as
autonomously replicating extra chromosomal DNA.
Plasmid
 Genetic element not contained within a chromosome.
 It occurs in many bacterial strains.
 Plasmids are circular DNA molecules that replicate independently of
the bacterial chromosome.
 They are not essential for the bacterium but may give it a selective
advantage.
 Some plasmids determine the production of proteins that can kill other
bacteria; others make bacteria resistant to antibiotics.
 Plasmids are extremely valuable tools in the fields of molecular biology
and genetics, specifically in the area of genetic engineering .
 Plasmids typically account for only a small fraction of a
bacterial genome corresponding roughly to a range
between 1 and 200 kilobase pairs.
 However, extremely large megaplasmids with sizes far
beyond 200kb have also been detected such as in
Rhizobium and others.
 Plasmids of more than 50 kb might be characterized as
large plasmids whereas plasmids used as tools in
molecular genetics are often smaller than 10 kb.
 Plasmid DNA can be separated from the rest of the bacteria
DNA because it is much smaller circles than the circular
bacterial chromosome.
 Most procedures involve cell lysis in which the bacterial
membrane is perforated and the contents released.
 In the process, the long circular chromosome become
sheared into linear fragments but the smaller plasmids
remain intact.
 Intact plasmid can be separated from linear DNA because
they remain supercoiled.
 The E. coli bacterial cells are resuspended in buffer
containing glucose, Tris, EDTA, and RNase.
 Glucose : gives osmotic shock that leads to the
rupture of cell wall and membrane.
 EDTA : stabilizes the cell membrane by binding
divalent cations (Mg++ and Ca++) and EDTA is an
important substance in all plasmid preparations
because it inhibits nuclease activity.
 Rnase : destroys the cell’s RNA.
 The resuspended cells are then treated with a SDS
and sodium hydroxide NaOH.
 SDS dissolves the phospholipid and protein
components of the cell membrane.
 The cell membrane lyses, releasing the cell
contents.
 Sodium hydroxide denatures both plasmid
and chromosomal DNA into single strands.
 Chromosomal DNA separates completely into
individual strands.
 The single-stranded plasmid loops remain linked
together like interlocked rings.
 Acidic potassium acetate forms an SDS/lipid/protein
precipitates.
 Acetic acid neutralizes the NaOH.
 At neutral pH, genomic DNA renatures and is trapped
in the SDS/lipid/protein precipitate.
 The plasmid DNA renatures into double-stranded
molecules that remain in solution.
Procedure:
Grow up a 3 ml overnight of E. coli broth media.
2. Pour 1.5 ml into an eppendrof tube and Pellet cells
by centrifuging at 12,000 rpm for 2 minutes.
3. Pour off supernatant and Add another 1.5ml of
culture to the same tube and centrifuge 2 min and
pour off the supernatent.
4. Resuspend the pellet by briefly vortex in 250µl of
solution I(stored at 4ºC ).
1.
5. Add 250 µl of solution II (stored at RT). Mix GENTLY
6.
7.
8.
9.
by inverting and rotating the tube several times. DO
NOT vortex. Put on ice 5 minutes.
Add 350 µl of cold solution III (stored at 4 º C).
Mix by inverting the tube 6-8 times. Put on ice for 5
minutes.
Spin 12 minutes at 12000 rpm in a microcentrifuge at
4º C. (Pellet contains: SDS, Lipids, Proteins and
Chromosomal DNA).
Transfer 350 µl of supernatant (contains plasmid
DNA). to a fresh tube.
10. Add 2 volumes ethanol at room tempreture. Mix and
stand for 2min at RT.
11. Pellet 5 min in microcentrifuge at RT. Dry, then
resuspend in 50 µl TE.
12. Analyze
your
preparations
using
agarose
gel
elctrophoresis by mix 8µl of the plasmid sample with
2µl of loading buffer or store at –20oC.
With Best
Wishes