Streptococcus mutans Transformation: Difference between revisions
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EricMiller (talk | contribs) (Created page with "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...") |
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Line 1: | Line 1: | ||
Day 1: | 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: | 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 8,000 grams | |||
##Pipette out 300uL of the supernatant into Virkon | |||
##Resuspend in 300uL of appropriate media | |||
##Second Wash | |||
##Spin them down for 1 minute at 5,000 grams | |||
##Dump out supernatant into Virkon | |||
##Resuspend in 300uL of appropriate media | |||
##Respuspension | |||
##Spin them down for 1 minute at 5,000 grams | |||
##Dump out supernatant into Virkon | |||
##Add in 300uL of appropriate media | |||
Adding XIP and DNA: | |||
##Set aside (-) tubes, nothing will be added to them | |||
##To achieve a final concentration of 2uM of XIP, add 6 uL of 100uM XIP will be added to each tube. | |||
##Add 300ng of DNA to each tube (with the exception of the (-)-s) 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. | |||
#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. | #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 | #Turn on Spectrometer |
Revision as of 16:46, 24 July 2023
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 8,000 grams
- Pipette out 300uL of the supernatant into Virkon
- Resuspend in 300uL of appropriate media
- Second Wash
- Spin them down for 1 minute at 5,000 grams
- Dump out supernatant into Virkon
- Resuspend in 300uL of appropriate media
- Respuspension
- Spin them down for 1 minute at 5,000 grams
- Dump out supernatant into Virkon
- Add in 300uL of appropriate media
Adding XIP and DNA:
- Set aside (-) tubes, nothing will be added to them
- To achieve a final concentration of 2uM of XIP, add 6 uL of 100uM XIP will be added to each tube.
- Add 300ng of DNA to each tube (with the exception of the (-)-s) 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.
- 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.