Streptococcus mutans Transformation

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Day 1: Streaking Out Plates

  1. Warm 1 BHI Plate
  2. Triple streak out S. mutans from freezer
  3. Allow overnight growth in a 37˚C 5% CO2 incubator

Day 2: Overnight Growth

  1. Add 1500uL of BHI to a well of the 96-well plate
  2. Add ~5 colonies of S. mutans to the BHI
  3. Allow overnight growth in a 37˚C 5% CO2 incubator

Day 3: Transformations

  1. Add 1500uL of SMUR to a well of the 96-well plate
    1. Have one well set aside for a negative control, no XIP or DNA to this well
  2. Pipette up and down the overnight culture with 500uL. Immediately after add 15uL of the overnight into SMUR wells.
  3. Place 96-well plates in 37˚C 5% CO2 incubator for 3.5 hours.
  4. Add 1000ng/mL of plasmid DNA to transformation condition
  5. Add 9.86 µL of 1521uM XIP to transformation condition (100uM).
  6. Allow to sit overnight for 14 hours.

Day 4: Plating Transformations

  1. Warm BHI plates and 1000ug/mL spectinomycin BHI plates
  2. Plate 200 uL of transformations/negative control on spectinomycin BHI plates
  3. Plate 100 uL of transformations diluted to 10^-2 on spectinomycin BHI plates
  4. Plate 100 uL of transformations diluted to 10^-6 on BHI plates
  5. Allow growth in a 37˚C 5% CO2 incubator for about ~48 hours.
  6. Count colonies and calculate transformation rates