Streptococcus mutans Transformation

Day 1:

1. 1. Label and add 3ml of BHI to two sterile test tubes
   2. Using a green inoculation loop, transfer 3 colonies of S. mutans
   3. Allow this culture to grow overnight in the 37˚C 5% CO2 incubator. The other BHI culture is for a future blank.

Day 2: Setting up

1. 1. Turn on the spectrophotometer
   2. Turn on the dry water bath, fill 8 of the divots with DI water and set to 37˚C.
   3. Get a sterile test tubes for each strain, and one for the blank.
   4. Fill the tubes
      • Strains to transform: 3 ml of BHI
      • Blank: 5-7ml of BHI
   5. Warm all of the tubes in the large divots in the 37˚C heat block; allow these to warm for at least 20 minutes.
   6. Place a mini-vortexer by the flame
   7. Prepare 500ml Virkon

Inoculation:

1. 1. After 20 minutes of warming, retrieve tubes.
   2. Grab overnight samples, vortex them and add 150uL (1:20 dilution) of the appropriate culture to the new tubes.
   3. Place these into the 37˚C 5% CO2 incubator. Allow them to grow for 2 hours and 30 minutes before checking them.
   4. After 2 hours and 30 minutes, blank the spectrophotometer with the blank, and vortex + wipe down the tubes and check the absorbance. If the absorbance is between 0.2 and 0.3 proceed; if not either allow to grow longer or dilute with warm BHI from the blank.

Washing the cells — BSL2 hoods:

1. 1. Label enough microtubules for conditions and replicates
   2. First wash:
      • Add 300 uL of the culture to these tubes
      • Spin them down for 2 minute at 8,000g
      • Pipette out 300uL of the supernatant into Virkon
      • Resuspend in 300uL of appropriate media
   3. Second Wash
      • Spin them down for 1 minute at 8,000g
      • Dump out supernatant into Virkon
      • Resuspend in 300uL of appropriate media
   4. Respuspension
      • Spin them down for 1 minute at 8,000g
      • Dump out supernatant into Virkon
      • Add in 300uL of appropriate media

Adding XIP and DNA:

1. 1. To achieve a final concentration of 2uM of XIP, add 6 uL of 100uM XIP will be added to each tube.
   2. Set aside negative control tubes, nothing more will be added to them
   3. Add 300ng of DNA to each tube (with the exception of the negative controls) and then vortex.
   4. Place all of the tubes in the 37˚C dry heat-block for 2.5 hours.
   5. Place appropriate number of 1000ug/mL Spectinomycin plates in the incubator, and appropriate number of BHI plates in the incubator.
   6. Label dilution tubes for appropriate strains and concentrations (for both transformation dilutions and dilutions of total cells)
      • Total cells: 10-5
      • Transformation dilutions: 10-1
   7. Allow all plates to grow for 36-48 hours in the 37˚C 5% CO2 incubator before counting colonies.