Transformation Preparation 1
Teacher Preparation - Transformation
You will need:
4 days before:
• 1 E. coli agar plate
• 15 Luria agar slants
2-3 days before:
• 2 bottles Luria Agar
• sterile water
• vial of ampicillin powder
• glass pipet and bulb
• 60 Petri plates
• marker
4 Days Before Lab:
1. Prepare the agar slants for class by inoculating with E.
coli bacteria.
a. Unwrap a sterile applicator swab.
b. Use it to collect some E. coli bacteria from the E. coli agar
plate.
c. Inoculate 15 Luria agar slants by gently swiping the swab
on the agar. Recap the agar slants and incubate at room
temperature for 4 days, or in a 37ºC incubator for 2 days,
until bacterial colonies are visible. Once colonies are
present, the slants can be stored in a 4oC refrigerator.
2-3 Days Before Lab:
2. Melt the agar
a. Melt 2 bottles of prepared agar in a boiling water bath. The
water level should cover the agar.
b. Loosen the cap so gases can escape, but do not remove
cap.
c. Swirl occasionally to ensure complete mixing.
3. Prepare the ampicillin solution
While the agar is melting,
a. Unwrap a glass pipet and attach bulb.
b. Add 2 ml of sterile water to the vial containing the
powdered ampicillin.
c. Cap the vial and shake vigorously.
d. Initial and date the ampicillin solution.
e. Keep ampicillin solution on ice.
Copyright © 2007 MassBioEd
Transformation Preparation 2
4. Cool agar
After the Luria agar is melted, place it in a 50ºC water bath
and use a thermometer taped to the outside of the bottle to
monitor the agar’s temperature. Be sure the agar has cooled
to 50ºC before proceeding to the next step.
5. Label the plates
a. While the agar is cooling, label the outside bottom of 30
plates “LB/Amp.” Write along the perimeter of the plate.
b. Label the bottom of 30 more plates “LB.”
6. Add ampicillin to one bottle of Luria agar
a. When the agar has cooled to 50ºC, add the entire 2ml of
the ampicillin solution to one of the bottles of Luria agar.
b. Swirl to mix.
c. Label the bottle “LB/Amp.”
7. Pour the plates
a. Line up 30 “LB/Amp” plates. Pour enough agar with
ampicillin to cover the surface of each plate. Lift the lid of
the plate just enough to admit the neck of the bottle. Avoid
touching the bottle to any surface.
b. Repeat with plain Luria agar in 30 “LB” plates.
c. Allow the agar to solidify in the Petri plates before
moving.
d. When the agar is solidified, store the plates upside down in
the refrigerator.
e. 30 minutes before use, take the plates out of the
refrigerator and store at room temperature.
Copyright © 2007 MassBioEd