Title: The Effect of LB, AMP, ARA, and pGlO on the E.Coli’s ability to grow or not.
Purpose: The purpose is to see the gene transformation in infected bacteria (E.Coli) added to the pGlO jellyfish gene.
1. If LB and (-)pGLO are added to E.Coli then, there will be max bacteria growth, and it will not glow.
2. If LB, Ampicillin and (-)pGLO are added to E.Coli then, there will be no bacterial growth, and it won’t glow.
3. If LB, ampiillin, and (+)pGLO are added E.Coli, then there will be max bacterial growth, and it will glow.
4. If LB, ampicillin, arabinose sugar, and (+)pGLO are added to E.Coli , then there will be max bacterial growth, and it will glow.
1. Sterilize work station
2. Label two microscopes one with (+)pGLO and another with (-)pGLO. Label names and then put on rack
3. Open tubes with sterile transfer pipet, transfer 250 microliters of transformation solution into each tube
4. Place tubes on ice
5. Use sterile loop to pick up single colony of bacteria form starter plate. Pick up (+)pGLO tube and immerse loop into the transformation tube at the bottom. Spin with index finger and thumb till clear. Place tube back in rack on ice, using new sterile loop. Repeat for (-)pGLO. 6. Examine pGlO DNA solution with the UV lamp. Note observation. Immerse new sterile loop into the pGlO plasmid DNA stock tube. Take out loopful. There should be a film of plasmid solution across the ring. This is similar to seeing a soapy film across a ring for blowing bubbles. Mix loopful into the cell suspension of the +pGLO tube. Close tube, and return it to the rack, on ice. Also close –pGLO tube. Don’t add plasmid to the –pGLO tube. Why not? 7. Incubate the tubes on ice for 10 minutes, and make sure there are pushed all the way down so there is no contact with the ice. 8. While the tubes are on ince, label your 4 LB nutrient agar plates on the bottom
Label one LB/amp plate: +pGlo
Label one LB/amp/ara plate: +pGLO
Label the other LB/amp plate: -pGLO
Label the LB plate: -pGLO
9. Using foam rack as holder, transfer both the (+) pGLO (-) pGLO tubes into water bath, set at 42 degrees Celsius, for 50 seconds. Makes sure tubes go all the way down and stick out so it touches warm water. When 50 seconds are done, place both tube back on rack. For better results, the transfer form ice (0 degrees Celsius) to 40 degrees Celsius and then back to the ice must be rapid. Incubates tubes on ice for 2 minutes.
10. Remove rack and place on bench top. Open a tube, using pipet, and add 250 ul of LB nutrient broth to the tube, and reclose it. Repeat with other tube. Incubate the tubes for 10 minutes.
11. Tap tubes with finger to mix. Using new pipet for each tube, pipet 100 ul of the transformation and control suspensions onto appropriate plates.
12. Spread suspensions around the surface of LB nutrient agar by quickly skating the flat surface of a new sterile loop back and forth. Do not press deep.
13. Stack up plates. Put your group name and period on bottom and place them upside down in the 37 degrees Celsius incubator. Observation:
Pglo Lesson 1
1. An organism better suited for total genetic transformation would be one composed of a single cell.
2. A better candidate for this investigation would be an organism in which each new generation develops and reproduces quickly.
3. The traits the organism should have is that they shouldn’t be aggressive, and be very calm.
4. The best choice for genetic transformation would be bacterium because they are calm and single celled.
Pglo Lesson 2
1. Plate number one will look most like the original because it doesn’t have ampicillin.
2. Plate numbers three and four will have E.Coli because the students added the Jellyfish DNA.
3. Plates one and four should be compared to determine if any genetic transformation has occurred because plate one has the original E.Coli, and plate four because…