Research Methods 2.5 Plasmid Purification

3 steps of plasmid purification

1. growing cells in a liquid culture - cells from isolated colony placed in LB broth via inoculation and only a single colony so all cells will have the same plasmid

2. purifiying the plasmid DNA from the culture

3. performing a restriction digest on the purified DNA to determine whether the DNA insert in the plasmid is the expected size

why is liquid culture used

E.coli needs nutrients, oxygen, and warmth for optimal growth

what does LB broth provide

nutrients composed of tryptone (peptides), yeast extract, and salt.

how is oxygenation provided to the cells

through shaking which is vital to growth

4 phases of bacterial growth

1. lag phase

2. exponential growth phase (cells are harvested in the late growth phase)

3. stationary phase (number of bacteria do not change)

4. death phase

what does purification do

separate plasmid DNA from genomic DNA

parts of purification

1. cell lysis and alkaline treatment of the lysate

2. neutralization

3. purification and concentration

cell lysis and alkaline treatment of lysate

bacteria must be lysed to release the cell contents but not too harsh. bacteria live in a dilute aqueous environment with a higher solute concentration inside the cell. lysing means disrupting the cell wall and cell membrane. alkaline solution contains NaOH, SDS, and EDTA. SDS disrupts cell membranes and denatures proteins. EDTA removes Mg2+ ions to destabilize cell wall and inactivate DNases. when the cell wall is weakened, the cell bursts

what happens under alkaline conditions

under pH (12-12.5) H bonds are broken, double helix unwinds, strands separate, and gDNA is denatured. plasmid DNA molecules are circular and supercoiled so the strands stay linked.

neutralization

high salt solution neutralizes alkaline pH of salt so the pH decreases and gDNA renatures and aggregates and high salt helps in precipitating out proteins. cellular debris including gDNA is removed by centrifugation. clarified lysate contain plasmid DNA.

purification and concentration

remove salts and other contaminants, concentrates DNA into a smaller volume, and column chromatography is used because DNA binds to the column in high concentrations of chaotropic salts.

what is added instead of ampicillin

carbanicillin because it is the stable form of ampicilin

aurum lysis solution

chemically breaks cells open, and solution has to be mixed by inverting because pipetting/vortexing can shear bacterial gDNA

what does the neutralization solution do

precipitates out gDNA, proteins, and cellular debris, leaving plasmid DNA in solution.

what does centrifuging do

centrifuging for 5 minutes purifies plasmid DNA from bacterial lysate and plasmid DNA ends up in supernatant

what does pipetting directly onto the column do

binds plasmid DNA to matrix