Streptococcus mutans Transformation: Difference between revisions

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Day 1:
Day 1: Streaking Out Plates
##Label and add 3ml of BHI to two sterile test tubes
# Warm 1 BHI Plate
##Using a green inoculation loop, transfer 3 colonies of ''S. mutans''
# Streak out S. mutans from freezer with inoculating loop in a BSL II hood.
##Allow this culture to grow overnight in the 37˚C 5% CO2 incubator. The other BHI culture is for a future blank.
# Allow overnight growth in a 37˚C 5% CO2 incubator.


Day 2: Overnight Growth
# Add 1000uL of SMUR to the number of wells you will need in a 96-well plate.
# Add 1 colony of S. mutans to each SMUR well.
# Allow overnight growth in a 37˚C 5% CO2 incubator
Day 3: Transformations
DO the following steps in a BSL II hood.
# Add 1000uL of SMUR to a new well of the 96-well plate for each transformation you plan to do. Allow these to warm at 37˚C for at least 30 minutes.
## Have one well set aside for a negative control, add no XIP or DNA to this well.
# Pipette up and down the overnight culture with a P1000 set 500uL. Immediately after add 10uL of the overnight into the new SMUR wells.
# Place 96-well plates in 37˚C *ambient* CO2 incubator for 3.5 hours.
# Add 300ng/mL of plasmid DNA to transformation condition
# Add 6.57 µL of 1521uM XIP to transformation condition (10uM of XIP).
# Place the 96-well plate in the  Ambient CO2 37˚C incubator overnight for 14-20 hours.


 
Day 4: Plating Transformations
Day 2:
# Warm BHI plates (for total cell count) and BHI plates with appropriate selection antibiotic.  
Setting up
# Plate 100 uL of undiluted  negative control (sample with no DNA or XIP) on an antibiotic BHI plate.
##Turn on the spectrophotometer
# Plate 100 uL of transformations diluted to 10^-2 on antiobiotic BHI plates
##Turn on the dry water bath, fill 8 of the divots with DI water and set to 37˚C.
## do 1:10 dilutions in PBS. Before Diluting, pipette transformation sample up/down vigourously with a P1000 set 500uL.
##Get a sterile test tubes for each strain, and one for the blank.
# Plate 100 uL of transformations diluted to 10^-5 on BHI plates
##Fill the tubes
## do 1:10 dilutions in PBS. Before Diluting, pipette transformation sample up/down vigourously with a P1000 set 500uL.
##*Strains to transform: 3 ml of BHI
# Allow growth in a 37˚C 5% CO2 incubator for about ~48 hours.
##*Blank: 5-7ml of BHI
##colonies will be visible after 24 hours, but much easier to count after 48 hours.
##Warm all of the tubes in the large divots in the 37˚C heat block; allow these to warm for at least 20 minutes.
# Count colonies and calculate transformation rates
##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.

Latest revision as of 15:51, 22 July 2024

Day 1: Streaking Out Plates

  1. Warm 1 BHI Plate
  2. Streak out S. mutans from freezer with inoculating loop in a BSL II hood.
  3. Allow overnight growth in a 37˚C 5% CO2 incubator.

Day 2: Overnight Growth

  1. Add 1000uL of SMUR to the number of wells you will need in a 96-well plate.
  2. Add 1 colony of S. mutans to each SMUR well.
  3. Allow overnight growth in a 37˚C 5% CO2 incubator

Day 3: Transformations DO the following steps in a BSL II hood.

  1. Add 1000uL of SMUR to a new well of the 96-well plate for each transformation you plan to do. Allow these to warm at 37˚C for at least 30 minutes.
    1. Have one well set aside for a negative control, add no XIP or DNA to this well.
  2. Pipette up and down the overnight culture with a P1000 set 500uL. Immediately after add 10uL of the overnight into the new SMUR wells.
  3. Place 96-well plates in 37˚C *ambient* CO2 incubator for 3.5 hours.
  4. Add 300ng/mL of plasmid DNA to transformation condition
  5. Add 6.57 µL of 1521uM XIP to transformation condition (10uM of XIP).
  6. Place the 96-well plate in the Ambient CO2 37˚C incubator overnight for 14-20 hours.

Day 4: Plating Transformations

  1. Warm BHI plates (for total cell count) and BHI plates with appropriate selection antibiotic.
  2. Plate 100 uL of undiluted negative control (sample with no DNA or XIP) on an antibiotic BHI plate.
  3. Plate 100 uL of transformations diluted to 10^-2 on antiobiotic BHI plates
    1. do 1:10 dilutions in PBS. Before Diluting, pipette transformation sample up/down vigourously with a P1000 set 500uL.
  4. Plate 100 uL of transformations diluted to 10^-5 on BHI plates
    1. do 1:10 dilutions in PBS. Before Diluting, pipette transformation sample up/down vigourously with a P1000 set 500uL.
  5. Allow growth in a 37˚C 5% CO2 incubator for about ~48 hours.
    1. colonies will be visible after 24 hours, but much easier to count after 48 hours.
  6. Count colonies and calculate transformation rates
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