User:Kalkao/notebook/Making Plaques
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- Obtain T-broth soft agar (0.7% agar)
- Place in the microwave for approximately 2 minutes and 45 seconds at 50% power
- Make sure the cap to the bottle is unscrewed and loose so that the bottle does not explode
- Place paper towels under the bottle to keep the microwave clean
- Place test tubes in the heat block set at level 5
- Place the soft agar in the 55*C incubator
- Obtain one 1.7mL centrifuge tube for every test tube in the heat block
- Add 250uL of overnight culture to each centrifuge tube
- Use fireboy to sterilize each pipette before placing in culture
- Flame each test tube opening before and after each use
- Obtain one pre-poured T-broth agar plate from the 4*C for every test tube in the heating block
- Obtain a lysate stock with phage
- Make several dilutions of the phage stock in T-broth to obtain a final dilution stock of approximately 1000 PFU/mL
- Obtain soft agar from incubator
- Pipette 3mL soft agar into each tube on the heat block
- Sterilize the pipette before placing in soft agar container
- Flame the test tube opening before and after each addition
- Pipette 2mL of the final diluted stock into a 2mL centrifuge tube
- Sterilize the pipette before placing in the dilution stock
- Sterilize the test tube before and after the pipette is inside
- Vortex the 2mL centrifuge tube
- Add phage from 2mL centrifuge tube to a tube with overnight culture
- Vortex the phage stock before extracting lysate
- Use a micropipette to obtain lysate (range from 10uL to 200uL to get between 100 to 300 phage per plate)
- After adding the phage, vortex the mixture
- Add the mixture to a test tube from the heat block
- Use a micropipette to suck up the whole mixture from the centrifuge tube
- When adding the mixture to the soft agar, pipette up and down to mix contents
- Flame the opening of the test tube before and after the addition
- Pour the phage/bacteria/soft agar mixture onto a plate
- Swirl the plate gently to ensure that the whole plate is covered evenly
- Do this quickly because the soft agar solidifies quickly
- Touch the lip of the tube to the agar to make sure all of the mixture leaves the test tube
- Place empty test tube in bleach bin, and place the cap in the white metal bin next to the sink
- Repeat steps 13 through 17 for every plate
- For blanks, use 250uL of distilled water to add to overnight culture tubes
- After agar has solidified, move the plates into the 37*C incubation room
- Plaques should form in approximately 4 hours