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Bacterial Transformation with ( pGLO Plasmid)

Bacterial Transformation with ( pGLO Plasmid). Lab #9: Molecular Biology. Purpose of this Lab. Learn how to insert a gene into bacteria (Heat Shock) Analyze how a gene can transform an organism and express that gene

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Bacterial Transformation with ( pGLO Plasmid)

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  1. Bacterial Transformation with (pGLO Plasmid) Lab #9: Molecular Biology

  2. Purpose of this Lab • Learn how to insert a gene into bacteria (Heat Shock) • Analyze how a gene can transform an organism and express that gene • Provide evidence that bacteria can take in foreign DNA in the form of a plasmid • Reinforce the following process: DNA  RNA  Protein  Trait • Observe how genes are regulated

  3. Applications of Genetic Transformation • Used in many areas of Biotechnology • Agriculture (pests, frost, & drought) • Bacteria (oil spills) • Gene therapy (sick cells into healthy cells) • Medicine (produce insulin & hormones)

  4. Key Terms to Know • DNA: Plasmid • Bacteria: E. coli (strain: HB101K-12) • Growth media: LB Broth (Luria & Bertani) • Ampicillin: Antibiotic kills bacteria “amp” • Arabinose: Sugar source for energy & carbon • Heat shock Process that increases permeability of the cell membrane to DNA • GFP: Green Fluorescent Protein (w/UV)

  5. The Genes of Interest • Ampicillin resistance • Gene regulation proteins-activate the GFP gene when arabinose is present • GFP: Green Fluorescent Protein -originally isolated from the jellyfish: Aequorea victoria

  6. Supplies for each Group of 4-5 (1) E. Coli-starter plate: has colonies present “LB” S (4) Agar plates: 1-LB 2-LB/amp 1-LB/amp/ara • Styrofoam holder tray w/ four vials (5) Pipets (7) Yellow inoculation loops: 1 package • Cup of crushed ice & water • Sharpie marking pen Masking tape

  7. Check your Materials-See List • Take out what you’ll need ***(leave packages sealed) • Label your capped vials as follows: • Yellow: “ - pGlo” • Orange: “ + pGlo” • Green: “TS”- Transformation Soln. • Pink: “LB broth”

  8. Using the Pipet Properly • Find the markings for each graduated volume on the pipet

  9. Basic Process -Begin with “Starter” colonies of E. coli -Place a colony into each of the two tubes provided. labeled: -pGlo and +pGlo (yellow loop) -Add the pGlo plasmid to the +pGlo tube only(yellow loop) -Place tubes on ice (10 min) bottom of tubes should be exposed to the ice -Label the four plates as indicated in your guide -Heat Shock the tubes in water bath (50 sec.) -Return to the ice (2 min) -Add 250 L of LB broth to both tubes (sterile pipette each time) -Add 100 L of tube contents to the appropriate plates -Spread the samples (w/yellow loop) & tape all four plates together. Be sure your period and group name is clearly indicated.

  10. Adding Transformation Solution

  11. Transferring Colonies, Labeling the Plates, & Using Heat Shock

  12. The Process of Heat Shock • Helps to increase the bacterial uptake of foreign DNA • Membrane becomes more permeable to DNA • Time is essential: -ice  water bath (42ºC) for 50 sec. ice • The number of transformants should increase

  13. Adding the Bacteria to the labeled Plates

  14. Expected Results

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