Plasmid DNA Isolation Exercise 8

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

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.

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

Plasmid Applications

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 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.

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.

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

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

Starting material

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 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

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 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.

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

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)‏

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

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

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.

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.