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Bioluminescent Genetic Transformation
Field Trip
Protocol
- Students (or teams) should label a screw-cap tube containing
bacteria with their initials.
- Using the disposable pipette, take up the DNA solution from
its tube and add it to the screw-cap tube. Screw the cap
back on and mix by gently shaking the tube.
- Place screw-cap tube on ice for 5 minutes.
- Heat shock the bacteria by placing the tube in a 42° C heat
block for 45 sec.
- Return the tube to the ice.
- Use the pipette to add growth media (LB Broth) from its tube
to the screw-cap tube.
- Incubate the screw-cap tube at room temperature for about 5
minutes.
- With a permanent marker, divide the bottom of the agar plate
in half.
- Label each plate with today’s date and the color
of the screw-cap.
- Students (or teams) should each label one half of the
plate with their initials (i.e. the plate is divided in
half and each half is initialed).
- Using the sterile inoculating loop, GENTLY transfer the
mixture from the tube onto the agar plate in an "S"
pattern.
- Incubate the plates at 31°C overnight to allow the bacteria
to grow.
Transformation Results
- Each table should have:
a) A 1.5 ml microcentrifuge tube marked LAR, which contains 50
ml 10X luciferin (1 mM final concentration) and 450 ml 100 mM
sodium citrate pH 5.5.
b) A plate of grown E. coli-luc bacteria.
- With the inoculating loop that was previously used, each student
should scrape some bacteria from the plate and CAREFULLY
add it to the LAR tube. Scrape and stir the bacteria into the
liquid in the tube to release it from the loop.
- Continue to harvest the bacteria in this until the tube is very
cloudy with bacteria. The more bacteria that is in the tube, the
brighter the light will be.
- After each student has added some bacteria, vigorously shake
the tube to break up the clumps of cells.
- View the tube in a dark place to see the bioluminescence.
Note: The disposable pipettes, inoculating loops, gloves,
bacterial tubes and plates used in this experiment should be disposed
of in biohazard trash.
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