K-12 Programs

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Bioluminescent Genetic Transformation Field Trip
Set-up

Materials/Equipment Needed

• Protocols- 1 per person
• Safety glasses- 1 per person
• Lab coats- 1 per person
• Gloves- 1 pair per person
• Plastic tube rack- 1 per bench
• Markers- 2 or more per bench
• Empty beaker (trash)- 1 per bench
• Beaker with crushed ice- 1 per bench
• E. coli tubes (yellow, green, orange and gray screw-cap tubes)- 1 tube per 2 students; Set up one color per bench
• DNA tubes (pink)- 1 tube per 2 students
• Growth medium tubes (yellow)- 1 tube per 2 students
• Disposable pipettes- 1 per 2 students
• Disposable inoculating loops- 1 loop per 2 students
• LB-Amp plates- 1 per 2 students
• LAR tubes- 1 tube per bench
• Grown E. coli-luc bacteria plates- 1 per 2 students (all 4 colors per lab)

The Day before the Lab

1. Make sure you have enough "grown" plates of each color (Y, G, YG, O) for each team of students. Leave the plates at room temperature in a drawer overnight.

IF NOT...

Label as many LB-Amp plates as necessary with the date and color (Y, G, GY, O) you are plating. Streak plates with bacteria from the frozen stock cultures or from stock plates in the refrigerator. Incubate plates at 31°C overnight.

2. Prepare one LAR tube per four students.

To a 1.5 ml microcentrifuge tube, add 450 µl of 100 mM sodium citrate, pH 5.5, and 50 µl of 10 mM luciferin (1 mM final concentration) into each tube. Label each tube LAR & store in the freezer.

3. Make sure you have enough LB Amp plates so that there is 1 plate per 2 students

IF NOT...

Order LB-Amp plates (1 plate/2 students) at least one week ahead.

4. Make sure you have enough E. coli tubes (yellow, green, orange and gray screw-cap tubes), DNA tubes (pink), and growth medium tubes (yellow).

   IF NOT...

   E.coli tubes-
   Label four 16 ml snap tubes and add 3 ml of LB Amp broth. Inoculate each tube with one of the luciferase clones from a stock culture or stock plate. Incubate the tubes overnight at 31°C. Pipette 100µl of the green culture into as many green screw-cap tubes as needed. Follow the same procedure for each of the other 3 colors (orange in orange, yellow in yellow, and yellow-green in gray.)

   DNA tubes-
   Pipette 200 µl of sterile de-ionized water into as many 0.65 ml pink microcentrifuge tubes as needed.

   Growth medium tubes-
   Pipette 200µl of LB broth into as many 0.65 ml yellow microcentrifuge tubes as needed.

The Day of the Lab

1. Set up as indicated under materials/equipment needed.
2. Put E.coli, LB broth and DNA tubes on ice.
3. Set out the grown E. coli-luc plates & LB-Amp agar plates on the front bench as well as the LAR tubes (allow them to thaw).

• Background
• Protocol
• Biotechnology Field Trips Home Page

Contact: Barbara Bielec, 608-273-9737