Team:British Columbia/Notebook/Protocols/plates

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Making Plates

Supplies:

Petri dishes

dH2O

Autoclavable container (500mL glass bottle)

Antibiotics (Stock solutions: Amp - 100mg/mL in 50% ethanol, Kan - 50mg/mL in H2O, Chl - 35mg/mL in 100%? ethanol, Tet - 15 mg/ml in 50% ethanol - all stored at -20°C)

Pipet gun + disposable 25mL pipet

Tinfoil (if using Tet)

LB Agar Powder

Colour scheme: Amp - red, Kan - blue, Chl - black, Tet - green

Steps:

1. Calculate amount of media needed (~20mL/plate).

2. Pour 400mL of diH2O into autoclavable container, make sure you remove any autoclave tape on the bottle.

3. Add LB Agar powder (check amount on stock container).

4. Mix as well as you can by swirling and gentle shaking.

5. Autoclave.

• Mark bottle with autoclave tape, make sure the lid is loose*

• Follow directions on autoclave*

• Set to 30 min*

• Before removing from autoclave make sure the pressure is at 0*

• Use heat-resistant gloves to remove from Autoclave*

6. Cool until you can hold your wrist against the bottle for 5 seconds without pain.

7. Add antibiotics (final concentrations: A-100-150μg/mL, K-50μg/mL, C-25μg/mL, Tet-10-15μg/mL); if using recommended stock solution concentrations, add 1μL of antibiotic stock per 1mL of media.

8. Swirl gently to mix antibiotics

• Steps 9-11 are to be done in the Biosafety cabinet in the biohazard room*
• Ensure the fan and light is on*
• Ensure no-one else is currently using the Biosafety cabinet*
• Ethanol everything (unopened plates package, Pipet gun, Pipets, Your arms up to the elbow, bottle of LB Agar) before bringing them inside the Biosafety cabinet*

9. Quickly pipet into plates.

• Avoid bubbles*

• Save the plate sleeve, don’t remove from cabinet, must remain sterile*

10. Leave to dry 20-30 min before using. Alternatively, leave to solidify 20-30 min before spread plating with appropriate volume of antibiotic.

11. For storage, place back in plate sleeve, label according to the colour scheme. If using a light-sensitive antibiotic (Tet), wrap in foil as well.

• Turn off the light and fan in the biohazard cabinet*

12. Keep at 4°C.

Notes:

Ampicillin degrades quickly, so don't make plates more than 2-3 weeks in advance and never leave them at 37°C for more than 16 hours.

Kanamycin can last 2-3 months. I don't know about chloramphenicol and tetracycline (not that stable, use like Amp), but they seem to be quite stable as well. Tetracycline is light sensitive.