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Zabulionis Unit Final Exam Notes

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Biology 2290F/G
Ray Zabulionis

ZABULIONIS UNIT Transformation Objective – Explain the biology/chemistry underlying the various steps of plasmid transformation 1. E. coli JM101 cells on pick plate 2. Add to cold calcium chloride  CaCl2is used to starve the DNA plasmid because there is no nutrient value  CaCl2also neutralizes charge of the phosphate group on the DNA, allowing for easy transportation with no repulsion (helps cell take up DNA, in simpler terms) 3. Ice bath 4. Divide into two test tubes – add TE, add plasmid  TE buffer = control o Essentially water, very weak buffer – used to dissolve DNA 5. Ice bath 6. 45°C heat pulse 7. Ice bath  Heat shock = helps bacteria to take up plasmid (by expansion and uptake)  Ice bath = immersion in ice helps keep the plasmid inside (with condensing of cellular contents) 8. Add LB broth  Lysogeny broth = food for bacteria  Provides bacteria with necessary nutrients and environment for optimal replication and growth 9. 37°C incubation  Also known as lag incubation  “Lag” because it takes time for bacteria to become resistant before plating 10.Plate and incubate overnight  LB (no amp) = control o Confluent growth – so much growth that individual colonies cannot be distinguished  LB + amp = control o Peptidoglycan – fibers that from cell wall o Ampicillin interferes with synthesis of new cell walls o Peptidoglycan must be cross-linked to form cell wall – ampicillin does not allow this, causing the cell to grow weaker and weaker and eventually burst open due to osmotic pressure  LB + amp + plasmid o Plasmid (pGREEN) contains amp resistance gene o Amp resistance gene codes for an enzyme which is able to destroy ampcilling Underlying Biology Objective – Explain the underlying biology for transformation, satellite colonies, antibiotic action and resistance of ampicillin, plasmid structure  Adhesion zones – where inner and outer membranes are fused to pores in the bacterial cell wall o Covered with lipopolysaccharide (LPS) difficult to pass through o Ice bath = allows DNA to pass without getting caught by fibers of the LPS o Heat pulse = causes thermoimbalance, giving a physical push o Calcium chloride = LPS, as well as DNA, have a negative charge; calcium chloride aids in neutralizing the charge of the DNA o Inner membrane has a resting cell negative potential which is maintained by a hydrogen pump – heat pulse causes this pump to stop working and the plasmid can now enter the cell  E. coli origin of replication is very controlled o Stringent – makes only one copy or a few copies, then shuts down  pGREEN origin of replication is not controlled o Relaxed – can make a large number of copies, only limiting factor is how much room is available in the cell  Two steps in the transformation process: 1. Competence – getting the plasmids in, cell is said to be competent if it can take in a plasmid  Competent cells = neutralized (done with calcium chloride) 2. Establishment – cell needs to recognize promoter on plasmid, transport protein and replicate  Only cells that are competent and established can form a transformant – it is possible for a cell to be competent but not undergo establishment  Satellite colonies – found surrounding the transformant o Tiny colonies that grow around an antibiotic resistant colony, which releases enzymes that degrade the antibiotic, benefitting the surrounding bacteria; satellite colonies themselves are not bacteria resistant  Ampicillin o Interferes with cell wall synthesis o Can only kill actively growing cells – when cell is dormant, ampicillin will have no effect  Importance of controls o Negative control – no growth is expected; to ensure that cells do not become resistant on their own without plasmid o Positive control – growth is expected; to ensure that cells can survive the protocol Micropipette Objective – Micropipette usage  P1000 – blue cap o Range: 200 μL – 1000 μL o Volume example: 050 = 500 μL 
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