User:Kalkao/notebook/Making Plaques


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