Streptococcus mutans Transformation: Difference between revisions
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Day 1: Streaking Out Plates | Day 1: Streaking Out Plates | ||
# Warm 1 BHI Plate | # Warm 1 BHI Plate | ||
# | # Streak out S. mutans from freezer with inoculating loop in a BSL II hood. | ||
# Allow overnight growth in a 37˚C 5% CO2 incubator | # Allow overnight growth in a 37˚C 5% CO2 incubator. | ||
Day 2: Overnight Growth | Day 2: Overnight Growth | ||
# Add | # Add 1000uL of SMUR to the number of wells you will need in a 96-well plate. | ||
# Add | # Add 1 colony of S. mutans to each SMUR well. | ||
# Allow overnight growth in a 37˚C 5% CO2 incubator | # Allow overnight growth in a 37˚C 5% CO2 incubator | ||
Day 3: Transformations | Day 3: Transformations | ||
# Add | DO the following steps in a BSL II hood. | ||
## Have one well set aside for a negative control, no XIP or DNA to this well | # Add 1000uL of SMUR to a new well of the 96-well plate for each transformation you plan to do. Allow these to warm at 37˚C for at least 30 minutes. | ||
# Pipette up and down the overnight culture with 500uL. Immediately after add | ## Have one well set aside for a negative control, add no XIP or DNA to this well. | ||
# Place 96-well plates in 37˚C | # Pipette up and down the overnight culture with a P1000 set 500uL. Immediately after add 10uL of the overnight into the new SMUR wells. | ||
# Place 96-well plates in 37˚C *ambient* CO2 incubator for 3.5 hours. | |||
# Add 300ng/mL of plasmid DNA to transformation condition | # Add 300ng/mL of plasmid DNA to transformation condition | ||
# Add | # Add 6.57 µL of 1521uM XIP to transformation condition (10uM of XIP). | ||
# | # Place the 96-well plate in the Ambient CO2 37˚C incubator overnight for 14-20 hours. | ||
Day 4: Plating Transformations | Day 4: Plating Transformations | ||
# Warm BHI plates and | # Warm BHI plates (for total cell count) and BHI plates with appropriate selection antibiotic. | ||
# Plate 100 uL of undiluted | # Plate 100 uL of undiluted negative control (sample with no DNA or XIP) on an antibiotic BHI plate. | ||
# Plate 100 uL of transformations diluted to 10^- | # Plate 100 uL of transformations diluted to 10^-2 on antiobiotic BHI plates | ||
## do 1:10 dilutions in PBS. Before Diluting, pipette transformation sample up/down vigourously with a P1000 set 500uL. | |||
# Plate 100 uL of transformations diluted to 10^-5 on BHI plates | # Plate 100 uL of transformations diluted to 10^-5 on BHI plates | ||
## do 1:10 dilutions in PBS. Before Diluting, pipette transformation sample up/down vigourously with a P1000 set 500uL. | |||
# Allow growth in a 37˚C 5% CO2 incubator for about ~48 hours. | # Allow growth in a 37˚C 5% CO2 incubator for about ~48 hours. | ||
##colonies will be visible after 24 hours, but much easier to count after 48 hours. | |||
# Count colonies and calculate transformation rates | # Count colonies and calculate transformation rates |
Latest revision as of 15:51, 22 July 2024
Day 1: Streaking Out Plates
- Warm 1 BHI Plate
- Streak out S. mutans from freezer with inoculating loop in a BSL II hood.
- Allow overnight growth in a 37˚C 5% CO2 incubator.
Day 2: Overnight Growth
- Add 1000uL of SMUR to the number of wells you will need in a 96-well plate.
- Add 1 colony of S. mutans to each SMUR well.
- Allow overnight growth in a 37˚C 5% CO2 incubator
Day 3: Transformations DO the following steps in a BSL II hood.
- Add 1000uL of SMUR to a new well of the 96-well plate for each transformation you plan to do. Allow these to warm at 37˚C for at least 30 minutes.
- Have one well set aside for a negative control, add no XIP or DNA to this well.
- Pipette up and down the overnight culture with a P1000 set 500uL. Immediately after add 10uL of the overnight into the new SMUR wells.
- Place 96-well plates in 37˚C *ambient* CO2 incubator for 3.5 hours.
- Add 300ng/mL of plasmid DNA to transformation condition
- Add 6.57 µL of 1521uM XIP to transformation condition (10uM of XIP).
- Place the 96-well plate in the Ambient CO2 37˚C incubator overnight for 14-20 hours.
Day 4: Plating Transformations
- Warm BHI plates (for total cell count) and BHI plates with appropriate selection antibiotic.
- Plate 100 uL of undiluted negative control (sample with no DNA or XIP) on an antibiotic BHI plate.
- Plate 100 uL of transformations diluted to 10^-2 on antiobiotic BHI plates
- do 1:10 dilutions in PBS. Before Diluting, pipette transformation sample up/down vigourously with a P1000 set 500uL.
- Plate 100 uL of transformations diluted to 10^-5 on BHI plates
- do 1:10 dilutions in PBS. Before Diluting, pipette transformation sample up/down vigourously with a P1000 set 500uL.
- Allow growth in a 37˚C 5% CO2 incubator for about ~48 hours.
- colonies will be visible after 24 hours, but much easier to count after 48 hours.
- Count colonies and calculate transformation rates