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pGLO Bacterial Transformation, Purification and SDS gel

pGLO Bacterial Transformation, Purification and SDS gel. Timeline Tranformation. Monday1/7 Lecture and discussion Tuesday 1/8 Transformation Laboratory Transform cells and spread plates Wednesday 1/9 (FIRE) Data Collection and Analysis Observe transformants and controls

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pGLO Bacterial Transformation, Purification and SDS gel

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  1. pGLO Bacterial Transformation, Purification and SDS gel

  2. Timeline Tranformation Monday1/7 Lecture and discussion Tuesday 1/8 Transformation Laboratory Transform cells and spread plates Wednesday 1/9 (FIRE) Data Collection and Analysis Observe transformants and controls Analyze and interpret results Inoculation Picking Colonies and Inoculating Cell Cultures HW Extension Activities Calculate transformation efficiency

  3. Timeline Purification Thursday 1/10 Purification Phase 1 Bacterial Concentration and Lysis Tuesday 1/15 Purification Phase 2 Removing Bacterial Debris Purification Phase 3 Protein Chromatography

  4. Timeline SDS Gel Thursday 1/17—Prepare Samples, Run gel, stain, destain (helper stay 7th) Friday 1/18—View Results, turn in lab Monday 1/21—post lab quiz, turn in lab

  5. Prep for Transformation Lab • 3-7 days prior to lab • Prepare agar plates, arabinose and ampicillan,(page 13-15) • Aliquot solutions (page 17) • 24-36 hr prior (page 16-17) • Rehydrate E. coli • Streak starter plates • Prepare pGLO plasmid

  6. Prep for Lab Purification • Before Inoculation Lab (Page 6-7) • Prepare liquid culture media, shaking incubator • Before Purification Phase 1 (page 8) • Rehydrate lyszyme • Set up centrifuge

  7. Prep. For SDS Gel lab check for supplies 95 water bath

  8. pGLO Bacterial Transformation Introduction to Transformation Transformation Laboratory Data Collection and Analysis Calculate Transformation Efficiency

  9. Background information • Genetic transformation: • Occurs when a cell takes up and expresses a new piece of genetic material-DNA • pGLO plasmid contains GFP gene and the gene for beta-lactamase, the ampicillin resistant gene • Transformed cells are found on the LB/amp and LB/amp/ara plates

  10. Lab Overview

  11. Transformation Tips • Transform cells and spread plates • Be careful when handling E. coli: It could cause food poisoning • Decontaminate work surfaces every day and after any spill • All contaminated liquid or solid wastes are decontaminated before disposal • All persons must wash their hands after handling bacteria and before exiting the lab • DO NOT mouth pipet, eat, or drink while in lab • Wear protective eyewear and gloves • Use a 10% bleach solution for at least 20 min for sterilization of loops and pipets • Observe transformants and controls/Analyze and interpret results • Observe the color and glow of the bacteria under a UV light • Fewer colonies of bacteria if amp negatively affected growth • Equal numbers of colonies on both LB nutrient agar and LB/amp agar if amp had no effect • The presence of any colonies on the amp plate would suggest that those bacteria are resistant to the antibiotic ampicillin

  12. Extension Activity • Calculate transformation efficiency • How to determine the extent to which genetic transformation of E. coli cells was successful • TIPS: • Transformation efficiency represents the total number of bacterial cells that express the green protein, divided by the amount of DNA used in the experiment • Transformation efficiency= Total number of colonies growing on agar plate Amount of DNA spread on the agar plate

  13. Green Fluorescent Protein Purification Genetic Transformation Review Inoculation- Growing a Cell Culture Purification Phase 1 Purification Phase 2 Purification Phase 3

  14. Background Information • pGLO plasmid contains GFP gene and the gene for beta-lactamase, the ampicillin resistant gene • When bacteria was plated onto LB agar containing arabinose (LB/amp/ara), GFP was expressed • When bacteria was plated onto LB agar that did not contain arabinose (LB/amp), the gene was turned off • Binding Wash Elution

  15. Chromatography Overview

  16. Purification Tips • Purification Phase 1Bacterial Concentration and Lysis • Centrifugation is a technique used to separate molecules on the basis of size by high speed spinning • Centrifugation results in a pellet of bacteria found at the bottom of the tube • The liqu • Purification Phase Removing Bacterial Debris • This final centrifugation step serves to separate the large particles of lysed bacteria (such as the cell membrane and walls) from the much smaller proteins, including GFP • The supernatant will fluoresce bright green upon exposure to UV light • Carefully remove the supernatant • id that resides above the pellet is called the supernatant

  17. Purification Tips • Purification Phase 3 Protein Chromatography • Bacteria contain thousands of endogenous proteins from which GFP must be separated • In chromatography, a cylinder, or column is densely filled with a “bed” of microscopic beads that will form a matrix through which proteins must pass before being collected • GFP “sticks” to the column, allowing it to be separated from the bacterial contaminants • Equilibration buffer Binding buffer Wash buffer Elution buffer

  18. GFP Protein on Polyacrylamide Gels

  19. Polyacrylamide gels • Tutorial 23 and 25 from disc.

  20. Background Information/Tips • Pages 2-8 • Only 4 stations (1, 3, 5, 7)

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