Streptococcus mutans Transformation
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Day 1:
- Label and add 3ml of BHI to two sterile test tubes
- Using a green inoculation loop, transfer 3 colonies of S. mutans
- 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
- Turn on the spectrophotometer
- Turn on the dry water bath, fill 8 of the divots with DI water and set to 37˚C.
- Get a sterile test tubes for each strain, and one for the blank.
- Fill the tubes
- Strains to transform: 3 ml of BHI
- Blank: 5-7ml of BHI
- Warm all of the tubes in the large divots in the 37˚C heat block; allow these to warm for at least 20 minutes.
- Place a mini-vortexer by the flame
- Prepare 500ml Virkon
Inoculation:
- After 20 minutes of warming, retrieve tubes.
- Grab overnight samples, vortex them and add 150uL (1:20 dilution) of the appropriate culture to the new tubes.
- Place these into the 37˚C 5% CO2 incubator. Allow them to grow for 2 hours and 30 minutes before checking them.
- 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:
- Label enough microtubules for conditions and replicates
- 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
- Second Wash
- Spin them down for 1 minute at 8,000g
- Dump out supernatant into Virkon
- Resuspend in 300uL of appropriate media
- 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:
- To achieve a final concentration of 2uM of XIP, add 6 uL of 100uM XIP will be added to each tube.
- Set aside negative control tubes, nothing more will be added to them
- Add 300ng of DNA to each tube (with the exception of the negative controls) and then vortex.
- Place all of the tubes in the 37˚C dry heat-block for 2.5 hours.
- Place appropriate number of 1000ug/mL Spectinomycin plates in the incubator, and appropriate number of BHI plates in the incubator.
- 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
- Allow all plates to grow for 36-48 hours in the 37˚C 5% CO2 incubator before counting colonies.