1. Plasmid DNA Isolation

2. Experiment Goals Extraction of plasmid DNA from E. Coli.
Analyze plasmid DNA by agarose gel electrophoresis and spectrophotometer.

3. 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.

4. 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.
F factor: An episome in bacterial cells that confers the ability to act as a genetic donor during conjugation
The Fertility factor (also known as F factor or sex factor) is a bacterial DNA sequence that allows a bacterium to produce a sex pilus necessary for conjugation

5. 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)

6. Plasmid Applications

8. 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.
Miniprep
Can be used to quickly find out whether the plasmid is correct in any of several bacterial clones. The yield is a small amount of impure plasmid DNA, which is sufficient for analysis by restriction digest and for some cloning techniques.
Maxiprep/bulkprep
Much larger volumes of bacterial suspension are grown from which a maxi-prep can be performed. Essentially this is a scaled-up miniprep followed by additional purification. This results in relatively large amounts (several micrograms) of very pure plasmid DNA.

9. 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 broth containing cells in a tube
Discard supernatant
LB: Luria Broth is used for maintenance and propagation of Escherichia coli. The E. coli grow faster in Luria Broth because the tryptone and yeast supply essential growth factors that the E. coli would otherwise have to synthesize. Luria Broth also contains essential electrolytes for transport and osmotic balance, due to the NaCl component.1

10. Plasmid DNA isolation Inactivation of Bacteria
Lysis of cells/ denaturation of DNA
Precipitation of DNA
Separate plasmid DNA from contaminants
Precipitation of Plasmid DNA
Precipitation of proteins
Precipitate Plasmid DNA

11. 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

12. 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

13. 3- Precipitation of DNA Centrifuge for 1 minute at high speed
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) potassium dodecyl sulfate (PDS)‏
Precipitate the genomic DNA
Centrifuge for 1 minute at high speed

14. 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

15. 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
Supernatant
Centrifuge
Incubate for 10 min. on ice
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.

16. 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
phenol, chloroform,
Centrifuge
Organic
Aqueous
phenol denatures proteins; chloroform denatures proteins and stabilizes the organic/aqueous boundary; isoamyl alcohol aids in the separation of the two phases and decreases the foaming

17. 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
Pellet
Centrifuge
Absolute ethanol & ammonium acetate
precipitated DNA
TE: 10 mM Tris-Cl, pH mM EDTA
• Wash pellet with 75% Ethanol (to remove salts)‏, & dry pellet
• Dissolve pellet with TE (or other aqueous solution)‏

18. 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