1. Plasmid DNA Isolation
Exercise 8

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

3. Plasmid Circular and double-stranded
Plasmids
A plasmid is an extrachromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the nuclear DNA.
E. coli
Chromosome
Circular and double-stranded
Plasmid size varies from 1 to over 100 kbp
The number of identical plasmids within a single cell can be zero, one, or even hundreds under some circumstances.

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., toluene or 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

6. 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)

7. Plasmid DNA isolation
Plasmid DNA isolation requires separation of this DNA from the chromosomal DNA in the bacterial cell as well as from the polysaccharides, lipids and proteins that constitute the cell.

8. Methods for Plasmid Isolation
There are several methods to isolate plasmid DNA from bacteria:
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 containing 20 mg/l ampicilin
Incubate overnight at 37oC
Centrifuge 1.5 ml of broth containing cells in a tube
Discard supernatant

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. Starting material

12. 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

13. 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

14. 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 (renatures 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

15. 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

16. 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.

17. 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

18. 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
• Wash pellet with 75% Ethanol (to remove salts)‏, & dry pellet
• Dissolve pellet with TE (or other aqueous solution)‏

19. 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

20. Assessing plasmid preparation
Verify by restriction digestion
Run undigested plasmid to see if it is mostly supercoiled
denatured
supercoiled

21. Analyzing Plasmids
After digestion, the restriction fragments are separated by gel electrophoresis.
The banding pattern from the restriction fragments provide a genetic “fingerprint” of the plasmid and gene insert.
restriction enzymes: BamHI and SacI.

22. Lane 1: Supercoiled (lower band) and open circular form (upper band)
Lane 2: Multimeric forms of supercoiled plasmid DNA Lane 3: Linearized form of a plasmid after restriction digestion with EcoRI. Lane 4: Sample contaminated with bacterial chromosomal DNA Lane 5: EcoRI digestion of a sample contaminated with bacterial genomic DNA which gives a smear above the plasmid DNA.