Streptococcus mutans Transformation

Day 1: Grow up a single colony of S. mutans (from BHI plate in fridge or incubator) in 3ml of of BHI, in 37 degree 5% CO2 incubator.

Day 2:

1. Warm two tubes of BHI to 37 degrees; one with 3ml (for bacteria growth), and one with ~7ml (as a blank and extra in case the OD600 of the experimental one needs to be diluted down) in either the incubator or heat block.
2. Turn on Spectrometer
3. 1:20 Dilution of overnight culture into the Exp BHI (from step 1) (150uL of the overnight culture) and place back into 37 degree 5% CO2 incubator. Leave undisturbed for at least 2 hours.

While the culture is incubating:

1. 1. Prepare 500ml of Virkon.
   2. Create 3ml of transformation media in the hood;
   3. Warm in the 37˚C 5% CO2 incubator until needed
2. Allow culture to grow for 2 hours uninterrupted and then check OD600 compared to blank. Before vortexing and checking OD, look at the tube in the light: is it cloudy at all? If not, it is not ready and put back into incubator with vortexing or checking absorbance.
3. If at absorbance between 0.13-0.2 proceed; if too much dilute down to 0.13 using pre-warmed BHI. BE SURE TO VORTEX. If not enough, wait another hour.
4. Put 300 uL of the OD600 ~ 0.1 EXP BHI into two 1.5ml microcentrifuge tubes (labelled: (-) and Experiment)
5. Centrifuge at 5,000 g/min for 1 minute

• • REMEMBER TO BALANCE THE CENTRIFUGE

1. Pipette the supernatant into virkon for each tube
2. Add 300uL of transformation media (or PBS) to each tube and vortex until cells are resuspended.
3. Centrifuge at 5,000 g/min for 1 minute
4. Repeat this washing one more time.
5. Resuspend in 300uL of transformation media
6. Add 6ul XIP (1521uM, this was written on the comS box. Some papers use 2uM as their final concentration for XIP? – let’s use a huge amount – 6ul will translate to a final concentration of ~30uM.)
7. Add in 300ng DNA, for a final concentration of 1ug/ml
8. Quick vortex
9. Place the tubes in 37˚ hot block (will warm up tubes faster) and incubate for 90 minutes.
10. After incubation, spin them down again at 5,000 g/min for 1 minute and pour off supernatant into virkon
11. Resuspend cells in 300uL of pre-warmed BHI and return to 37 degree hot block for another 90 minute incubation
12. Plate 30ul of the transformants onto 1000ug/ml spec BHI plates (pre-warmed to room temp.) with 200ul BHI and 5-7 beads. Distribute by the beads, allow to dry, and grow overnight in the 37˚ 5% CO2 incubator.
13. Plate xxil of the transformants onto no-antibiotic BHI plates (pre-warmed to room temp.) with 200ul BHI and 5-7 beads. Distribute by the beads, allow to dry, and grow overnight in the 37˚ 5% CO2 incubator.
14. Colonies will appear 24-36 hours later.