PGLO Plasmid Transformation and Analysis

  1. Label 2 sterile microfuge tubes: + p - p 2. Add 250µl of ice cold CaCl2 (calcium chloride) solution to each tube. 3. Obtain a streak plate of E. coli from the instructor. 4. Using a sterile loop, pick 4 - 5 isolated colonies from the plate, then add them directly to the cold CaCl2 solution in the +p tube. Twist the toothpick vigorously to dislodge the cells from the loop. Repeat for the –p tube. NOTE: Try to avoid scraping up agar when you are picking colonies. 5. Be sure that the cells are completely re-suspended in each tube by tapping and mixing. Note: You should not have chunks of colonies floating around 6. To the tube labeled “+ p” add 5 µl of your selected plasmid DNA from Part 1. I have added 5 µl of your selected plasmid DNA from Part 1 To the tube labeled “- p” as well by mistake (It is good for the discussion) Note: Be sure to add the plasmid directly to the cell suspension. 7. Incubate both tubes on ice for 10 minutes. While the tubes are sitting on ice, label your four LB nutrient agar plates on the bottom: • Label one LB/amp plate: + p • Label the LB/amp/ara plate: + p • Label the other LB/amp plate: - p • Label the LB plate: - p 8. Obtain your tubes from the ice and place both tubes at 42°C for exactly 50 seconds 9. Return both tubes immediately to the ice bucket for two minutes. 10. Add 250µl Luria Broth (“LB broth”) to each tube and mix. Be sure to change tips in between tubes. 11. Incubate the cells for 10 minutes at room temperature. 12. After your incubation is complete, you are ready to spread your cells onto the plate. 13. Add 100µl of your “– p” transformation mixture to each of the appropriate plates. Spread each plate one at a time with a sterile spreader. Your instructor will demonstrate this technique to you. 14. Repeat the process for your “+ p” transformation mixture. 15. Once you have spread your transformation mixtures on all 4 plates, let the agar plates sit on the bench for ~5 minutes. 16. Stack the agar plates lid side down and secure with paper tape containing your group’s initials. Place the plates in the 37°C dry incubator overnight. 17. You will have to come and check on your bacteria within 48 hours using the fluorescent light Post Lab (it attached to the order) Analyze your results in 1-2 paragraphs. Be sure to consult references to explain your data. Your response should include: the impact of your plasmid on your transformation; whether you selected pGLO or pRFP based on the electrophoresis results; why some plates glowed and others did not based on the growth environment; if the color of your fluorescene matched your expectation-why or why not.