TITLE: TAP 0.5%-0.75% impranil agarose plates
AUTHORS: Joao Vitor Molino, Barbara Saucedo

[DESCRIPTION]
The protocol describes the preparation of TAP media plates containing 0.5% to 0.75% Impranil, with the inclusion of antifungal and ampicillin. Notably, agarose was employed instead of agar to achieve a lighter gel color. These specialized plates serve the purpose of identifying Chlamydomonas reinhardtii strains secreting the plastic-degrading enzyme PHL7, discerned by the halo formation around the colonies.

[GUIDELINES]
All steps described in this protocol are intended to be conducted in a research laboratory.  Follow aseptic procedures

To keep plates from getting contaminated always use sterile technique

Shelf life 6 months

[STEPS]
SECTION: TAP media
1. Take a stir rod and place into a 1 L erlenmeyer flask then fill with 900 mL of Milli-Q water. Place flask on stir plate and stir gently.

SECTION: TAP media
2. Add 20 mL of 1M Tris Base
Add 1 mL of Phosphate Buffer II
Add 10 mL of Solution A
Add 1 mL of Trace Elements
Let it stir.
SECTION: TAP media
3. Place a pH probe into the TAP media solution and adjust to pH 7 with Glacial Acetic Acid. *Slowly add 1 mL-1.5 mL.

SECTION: TAP media
4. Measure out 15g of agarose and add it to the TAP media solution to have 1.5 % (v/v) per 1 L. Stir gently.

5. Top off TAP media with Milli-Q water until you get to 1 L, let it stir for a couple of minutes then remove it from the stir plate. Place a square foil paper on top of the flask to cover and label the flask. It is now ready to be autoclaved.

SECTION: Autoclave
6. Securely place the flask inside a suitable autoclavable container. Add water to the container, ensuring sufficient coverage around the flask. Load the autoclave with the container and set the autoclave parameters according to the manufacturer's guidelines. Use a cycle time of 01:00:00 (1 hour) with settings appropriate for your specific autoclave model (e.g., gravity 30). Start the autoclave cycle, following standard operating procedures for your laboratory's equipment.

Note: Refer to the autoclave manufacturer's instructions for precise settings and safety guidelines. Adjust time and settings as needed for your specific autoclave.

SECTION: Water bath cool down and sterile technique to pour plates
7. Once the autoclave has completed, take out the flask, place on stir plate to give a quick stir then place in a water bath at a temperature of 55 °C for 120 min - 240 min before adding antibiotics.

SECTION: Water bath cool down and sterile technique to pour plates
8. Place flask on stir plate and measure the temperature of the TAP media with an infrared thermometer. After the temperature is below 50 °C, using sterile technique, add the antibiotics and the rest of the ingredients. If temperature is still high, let it sit on the stir plate and cool down for a few more minutes. For 1 L of TAP media add 11 mL of 1.0302 mg/mL antifungal, 1 mL of ampicillin 150 mg/mL in 70 % (v/v) ethanol and either 5.0 mL or 7.5 mL of impranil (Anionic aliphatic polyester-polyurethane dispersion. Impranil DLN SD) for 0.5 % (v/v) or 0.75 % (v/v) of impranil per 1 liter of media. Let it stir.

SECTION: Water bath cool down and sterile technique to pour plates
9. As the media is stirring, set up the petri dishes on a clean flat surface near a flame and you are ready to pour.
SECTION: Water bath cool down and sterile technique to pour plates
10. Label and leave petri dish to dry overnight.