Team:British Columbia/Notebook/Protocols/plates
From 2014.igem.org
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<li> Mix as well as you can by swirling and gentle shaking.</li> | <li> Mix as well as you can by swirling and gentle shaking.</li> | ||
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<li> 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.</li> | <li> 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.</li> | ||
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Latest revision as of 00:19, 18 October 2014
>
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
- Calculate amount of media needed (~20mL/plate).
- Pour 400mL of diH2O into autoclavable container, make sure you remove any autoclave tape on the bottle.
- Add LB Agar powder (check amount on stock container).
- Mix as well as you can by swirling and gentle shaking.
- 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
- Cool until you can hold your wrist against the bottle for 5 seconds without pain.
- 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.
- 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
- Quickly pipet into plates:
- Avoid bubbles.
- Save the plate sleeve, don’t remove from cabinet, must remain sterile.
- Leave to dry 20-30 min before using. Alternatively, leave to solidify 20-30 min before spread plating with appropriate volume of antibiotic.
- 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
- Keep at 4°C.