Streptococcus mutans Transformation
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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:
- 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.
- Turn on Spectrometer
- 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:
- Prepare 500ml of Virkon.
- Create 3ml of transformation media in the hood;
- Warm in the 37˚C 5% CO2 incubator until needed
- 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.
- 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.
- Put 300 uL of the OD600 ~ 0.1 EXP BHI into two 1.5ml microcentrifuge tubes (labelled: (-) and Experiment)
- Centrifuge at 5,000 g/min for 1 minute
- REMEMBER TO BALANCE THE CENTRIFUGE
- Pipette the supernatant into virkon for each tube
- Add 300uL of transformation media (or PBS) to each tube and vortex until cells are resuspended.
- Centrifuge at 5,000 g/min for 1 minute
- Repeat this washing one more time.
- Resuspend in 300uL of transformation media
- 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.)
- Add in 300ng DNA, for a final concentration of 1ug/ml
- Quick vortex
- Place the tubes in 37˚ hot block (will warm up tubes faster) and incubate for 90 minutes.
- After incubation, spin them down again at 5,000 g/min for 1 minute and pour off supernatant into virkon
- Resuspend cells in 300uL of pre-warmed BHI and return to 37 degree hot block for another 90 minute incubation
- 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.
- 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.
- Colonies will appear 24-36 hours later.