Streptococcus mutans Transformation: Difference between revisions
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## Have one well set aside for a negative control, no XIP or DNA to this well | ## Have one well set aside for a negative control, no XIP or DNA to this well | ||
# Pipette up and down the overnight culture with 500uL. Immediately after add 15uL of the overnight into SMUR wells. | # Pipette up and down the overnight culture with 500uL. Immediately after add 15uL of the overnight into SMUR wells. | ||
# Place 96-well plates in 37˚C 5% CO2 incubator for 3.5 hours. | |||
# Add 1000ng/mL of plasmid DNA to transformation condition | |||
# Add 9.86 µL of 1521uM XIP to transformation condition (100uM). | |||
# Allow to sit overnight for 14 hours. | |||
Day 4: Plating Transformations | Day 4: Plating Transformations | ||
# Warm BHI plates and 1000ug/mL spectinomycin BHI plates | |||
# Plate 200 uL of transformations/negative control on spectinomycin BHI plates | |||
# Plate 100 uL of transformations diluted to 10^-2 on spectinomycin BHI plates | |||
# Plate 100 uL of transformations diluted to 10^-6 on BHI plates | |||
# Allow growth in a 37˚C 5% CO2 incubator for about ~48 hours. | |||
# Count colonies and calculate transformation rates |
Revision as of 14:38, 15 May 2024
Day 1: Streaking Out Plates
- Warm 1 BHI Plate
- Triple streak out S. mutans from freezer
- Allow overnight growth in a 37˚C 5% CO2 incubator
Day 2: Overnight Growth
- Add 1500uL of BHI to a well of the 96-well plate
- Add ~5 colonies of S. mutans to the BHI
- Allow overnight growth in a 37˚C 5% CO2 incubator
Day 3: Transformations
- Add 1500uL of SMUR to a well of the 96-well plate
- Have one well set aside for a negative control, no XIP or DNA to this well
- Pipette up and down the overnight culture with 500uL. Immediately after add 15uL of the overnight into SMUR wells.
- Place 96-well plates in 37˚C 5% CO2 incubator for 3.5 hours.
- Add 1000ng/mL of plasmid DNA to transformation condition
- Add 9.86 µL of 1521uM XIP to transformation condition (100uM).
- Allow to sit overnight for 14 hours.
Day 4: Plating Transformations
- Warm BHI plates and 1000ug/mL spectinomycin BHI plates
- Plate 200 uL of transformations/negative control on spectinomycin BHI plates
- Plate 100 uL of transformations diluted to 10^-2 on spectinomycin BHI plates
- Plate 100 uL of transformations diluted to 10^-6 on BHI plates
- Allow growth in a 37˚C 5% CO2 incubator for about ~48 hours.
- Count colonies and calculate transformation rates