Pouring the Plaques: Difference between revisions
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Xiwei Huang (talk | contribs) (Created page with "The top layer will consist of an equally sized layer made of the same broth and soft agar (.8%) mixed with 30 uL of bacterial culture and 100 uL of phage dilution 1. To prepare for pouring the phage layer: a. Prepare all phage pipettes b. Prepare filtered tips for phage c. Check that your dilutions are represent and organized d. Check that you collected all your warmed plates from the incubator e. Aliquot 4 mL of soft agar into your warming glass tubes f. Place b...") |
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1. To prepare for pouring the phage layer: | 1. To prepare for pouring the phage layer: | ||
a. Prepare all phage pipettes | a. Prepare all phage pipettes | ||
b. Prepare filtered tips for phage | |||
b. Prepare filtered tips for phage | c. Check that your dilutions are represent and organized | ||
d. Check that you collected all your warmed plates from the incubator | |||
c. Check that your dilutions are represent and organized | e. Aliquot 4 mL of soft agar into your warming glass tubes | ||
f. Place big soft agar bottle on styrofoam to prevent rapid heat diffusion | |||
d. Check that you collected all your warmed plates from the incubator | g. Make sure your vortexer is plugged in and the outlet is switched on | ||
e. Aliquot 4 mL of soft agar into your warming glass tubes | |||
f. Place big soft agar bottle on styrofoam to prevent rapid heat diffusion | |||
g. Make sure your vortexer is plugged in and the outlet is switched on | |||
2. Quickly but carefully (1 by 1): | 2. Quickly but carefully (1 by 1): | ||
a. Pipette | a. Pipette 100 uL of phage into the same glass tube | ||
b. Vortex your phage tube to confirm homogenous mixture | |||
b. Vortex your phage tube to confirm homogenous mixture | c. Pipette 30 uL of bacterial culture into a glass tube | ||
d. Vortex for about 5 seconds ( look for the tornado) | |||
c. Pipette | e. Open pre-labeled plate and pour mixture onto the LB layer | ||
f. Swirl gently to allow soft agar to spread evenly, cover, then set aside | |||
d. Vortex for about 5 seconds ( look for the tornado) | g. Allow about 15 seconds for a poured plate to set before moving | ||
e. Open pre-labeled plate and pour mixture onto the LB layer | |||
f. Swirl gently to allow soft agar to spread evenly, cover, then set aside | |||
g. Allow about 15 seconds for a poured plate to set before moving | |||
3. Repeat for desired number of plates | 3. Repeat for desired number of plates | ||
4. Incubate overnight at 37C with ambient CO2 | 4. Incubate overnight at 37C with ambient CO2 | ||
Latest revision as of 13:43, 19 March 2023
The top layer will consist of an equally sized layer made of the same broth and soft agar (.8%) mixed with 30 uL of bacterial culture and 100 uL of phage dilution
1. To prepare for pouring the phage layer:
a. Prepare all phage pipettes b. Prepare filtered tips for phage c. Check that your dilutions are represent and organized d. Check that you collected all your warmed plates from the incubator e. Aliquot 4 mL of soft agar into your warming glass tubes f. Place big soft agar bottle on styrofoam to prevent rapid heat diffusion g. Make sure your vortexer is plugged in and the outlet is switched on
2. Quickly but carefully (1 by 1):
a. Pipette 100 uL of phage into the same glass tube b. Vortex your phage tube to confirm homogenous mixture c. Pipette 30 uL of bacterial culture into a glass tube d. Vortex for about 5 seconds ( look for the tornado) e. Open pre-labeled plate and pour mixture onto the LB layer f. Swirl gently to allow soft agar to spread evenly, cover, then set aside g. Allow about 15 seconds for a poured plate to set before moving
3. Repeat for desired number of plates
4. Incubate overnight at 37C with ambient CO2