Question

Procedures pGLO Bacterial Plates Bacteria can be grown in liquid media (liquid culture) or on a solid substrate (plate culture). For the first experiment, you will be adding nothing (control), D-arabinose or L-arabinose to each plate. After letting the plates sit at room temperature for 10 minutes, you will add the bacteria to the plate using an inoculating loop and incubate the plates overnight at 37°C. Students will then look at each plate under a UV light to determine which, if any, of the sugars were able to activate transcription of the GFP gene. Protocol: 1. Add 100 uls of bacteria (competent cells produced previously) to each of 4 Eppendorf tubes. 2. To tubes 1 and 2 add 1ul of H?0. To tubes 3 and 4 add 1 ul of pGLO DNA. Be sure the 1ul gets into the tube! 3. Incubate all samples on ice for 15 minutes. 4. Place all samples at 42°C for 90 seconds 5. Place samples on ice for 2 minutes 6. Add tubes 1-4 to plates 1-4 (plate 1 LB, plate 2 LB/amp, plate 3 LB/amp + 5% D-arabinose, plate 4 LB/amp + 5% L-arabinose). 7. Incubate plates overnight at 37°C. 8. Determine which plates had bacterial growth, which had individual colonies and which were green. Questions: 1. What type of growth would you expect on each plate? Why? 2. Do some of the bacterial plates glow green and others don't? Why? 3. If you had bacteria that did not glow, could you do something to get them to start? If so, how?

          Procedures

pGLO Bacterial Plates
Bacteria can be grown in liquid media (liquid culture) or on a solid substrate (plate culture). For the first experiment, you will be adding nothing (control), D-arabinose or L-arabinose to each plate. After letting the plates sit at room temperature for 10 minutes, you will add the bacteria to the plate using an inoculating loop and incubate the plates overnight at 37°C. Students will then look at each plate under a UV light to determine which, if any, of the sugars were able to activate transcription of the GFP gene.

Protocol:

1. Add 100 uls of bacteria (competent cells produced previously) to each of 4 Eppendorf tubes.
2. To tubes 1 and 2 add 1ul of H?0. To tubes 3 and 4 add 1 ul of pGLO DNA. Be sure the 1ul gets into the tube!
3. Incubate all samples on ice for 15 minutes.
4. Place all samples at 42°C for 90 seconds
5. Place samples on ice for 2 minutes
6. Add tubes 1-4 to plates 1-4 (plate 1 LB, plate 2 LB/amp, plate 3 LB/amp + 5% D-arabinose, plate 4 LB/amp + 5% L-arabinose).
7. Incubate plates overnight at 37°C.
8. Determine which plates had bacterial growth, which had individual colonies and which were green.

Questions:

1. What type of growth would you expect on each plate? Why?
2. Do some of the bacterial plates glow green and others don't? Why?
3. If you had bacteria that did not glow, could you do something to get them to start? If so, how?

Procedures

pGLO Bacterial Plates
Bacteria can be grown in liquid media (liquid culture) or on a solid substrate (plate culture). For the first experiment, you will be adding nothing (control), D-arabinose or L-arabinose to each plate. After letting the plates sit at room temperature for 10 minutes, you will add the bacteria to the plate using an inoculating loop and incubate the plates overnight at 37°C. Students will then look at each plate under a UV light to determine which, if any, of the sugars were able to activate transcription of the GFP gene.

Protocol:

1. Add 100 uls of bacteria (competent cells produced previously) to each of 4 Eppendorf tubes.
2. To tubes 1 and 2 add 1ul of H?0. To tubes 3 and 4 add 1 ul of pGLO DNA. Be sure the 1ul gets into the tube!
3. Incubate all samples on ice for 15 minutes.
4. Place all samples at 42°C for 90 seconds
5. Place samples on ice for 2 minutes
6. Add tubes 1-4 to plates 1-4 (plate 1 LB, plate 2 LB/amp, plate 3 LB/amp + 5% D-arabinose, plate 4 LB/amp + 5% L-arabinose).
7. Incubate plates overnight at 37°C.
8. Determine which plates had bacterial growth, which had individual colonies and which were green.

Questions:

1. What type of growth would you expect on each plate? Why?
2. Do some of the bacterial plates glow green and others don't? Why?
3. If you had bacteria that did not glow, could you do something to get them to start? If so, how?

Added by Joshua R.

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