Pre-Laboratory Preparation |
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- Prepare the tetracycline stock solution.
- Open the capsule containing 250 mg
tetracycline-HCl and empty contents into the
bottle containing 40 mL denatured 50% ethanol.
- To mix, shake gently; label the bottle "Tetracycline
HCl Solution." Store in a freezer until needed.
- Prepare media plates.
(2 nutrient agar and 2 nutrient agar with tetracycline
for each team of four students.)
Melting Prepared Media: Always use aseptic
technique. Melt prepared media in a water bath and
pour into petri plates as follows:
- Remove cap seals and loosen caps on bottles.
- Place bottle(s) in large Pyrex beaker or saucepan.
Pour water into that container until it rises above
the level of the medium in the bottle.
- Heat the water on a hotplate until it boils.
- Boil gently for several minutes until the medium
is completely liquified. Test by swirling the bottles
gently. Caution! Use insulated gloves when handling hot materials.
- Turn off the heat and allow the temperature in the
water bath to drop to 45-50oC.
Pouring Media Plates: Pour 14 plates (requires two
bottles of agar).
- Line up petri plates in work area.
- Remove the cap from one bottle and flame the
mouth of the bottle.
- Lift the lid of the petri plate just enough to admit
the neck of the bottle. Avoid touching the neck of
the bottle to any surface.
- Pour the first plate by holding the top of the petri
plate in one hand at a 45o angle, while pouring the
liquified agar (Figure 1). Use enough media to
cover slightly more than the bottom of the plate
(about 15 mL). Replace the lid.
- Gently swirl the plate to evenly distribute the agar.
- Flame the mouth of the bottle immediately and
continue pouring plates as required. The agar will
solidify in 15 to 30 minutes. Do not move the plates until the agar has solidified. Following solidification, store agar plates upside down in the refrigerater until needed. Mark a "P" on the bottom of each nutrient agar plate.
Figure 1
Pouring tetracycline agar plates: Pour 14 tetracycline agar plates (requires
two bottles of nutrient agar and tetracycline stock solution).
- Add 0.25 mL of previously prepared tetracycline stock solution to each of the two bottles of
melted/liquified nutrient agar using aseptic
technique. Seal and gently swirl each of the bottles
to distribute the antibiotic evenly.
- Follow the procedure outlined above for pouring
media plates.
- After the agar has solidified, mark a "T" on the
bottom of each plate. Store plates upside down in a refrigerator until needed. The tetracycline in the agar plates is sensitive to light, so keep
poured plates away from light as much as possible.
Preparing nutrient broth: Eight tubes of sterile nutrientbroth are provided in this kit: 7 tubes (14 mL) and 1tube (15 mL). The 14 mL tubes will be inoculated with bacteria.
- Prepare inoculated cultures.
Culture Establishment (Breakout): Inoculate one
rehydration ("break out") media tube with the lyophilized E. coli as outlined
on the instruction card accompanying the culture.
Growing the bacteria: The bacteria must be in an active growth phase for transformation to take place. This "acceptance state" is termed competence. Manipulating bacteria to become competent must be done 24 hours prior to conducting the experiment.
- Grow bacteria in a broth media (tryptic soy or nutrient broth for E. coli strains) for 24 hours at 37oC. The broth will be turbid following this incubation period.
- On the day of the experiment, transfer 1 mL of the actively-growing culture to a fresh tube of nutrient broth. Transfer one tube for every two students or group of students.
- Incubate these new cultures (step 2) at 37oC, 4 to 6 hours before beginning the transformation protocol. This Timing is Critical for the Success
of the Transformation Experiment!
Plasmid Rehydration: Add 1 mL sterile water to lyophilized pBR322. Allow to stand undisturbed for one hour or rehydrate the day before needed for use and refrigerate overnight.
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AE Partners Collection Index
