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

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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