Streptococcus mutans Transformation: Difference between revisions

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(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...")
 
(updating protocol)
 
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Day 1:
Day 1: Streaking Out Plates
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.
# Warm 1 BHI Plate
# Streak out S. mutans from freezer with inoculating loop in a BSL II hood.
# Allow overnight growth in a 37˚C 5% CO2 incubator.


Day 2:
Day 2: Overnight Growth
#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.
# Add 1000uL of SMUR to the number of wells you will need in a 96-well plate.
#Turn on Spectrometer
# Add 1 colony of S. mutans to each SMUR well.
#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.
# Allow overnight growth in a 37˚C 5% CO2 incubator
While the culture is incubating:
##Prepare 500ml of Virkon.  
Day 3: Transformations
##Create 3ml of transformation media in the hood;
DO the following steps in a BSL II hood.  
##Warm in the 37˚C 5% CO2 incubator until needed
# 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.
#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.
## Have one well set aside for a negative control, add no XIP or DNA to this well.  
#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.
# Pipette up and down the overnight culture with a P1000 set 500uL. Immediately after add 10uL of the overnight into the new SMUR wells.
#Put 300 uL of the OD600 ~ 0.1 EXP BHI into two 1.5ml microcentrifuge tubes (labelled: (-) and Experiment)
# Place 96-well plates in 37˚C *ambient* CO2 incubator for 3.5 hours.
#Centrifuge at 5,000 g/min for 1 minute
# Add 300ng/mL of plasmid DNA to transformation condition
**REMEMBER TO BALANCE THE CENTRIFUGE
# Add 6.57 µL of 1521uM XIP to transformation condition (10uM of XIP).
#Pipette the supernatant into virkon for each tube
# Place the 96-well plate in the  Ambient CO2 37˚C incubator overnight for 14-20 hours.
#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
Day 4: Plating Transformations
#Repeat this washing one more time.
# Warm BHI plates (for total cell count) and BHI plates with appropriate selection antibiotic.  
#Resuspend in 300uL of transformation media
# Plate 100 uL of undiluted  negative control (sample with no DNA or XIP) on an antibiotic BHI plate.
#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.)
# Plate 100 uL of transformations diluted to 10^-2 on antiobiotic BHI plates
#Add in 300ng DNA, for a final concentration of 1ug/ml
## do 1:10 dilutions in PBS. Before Diluting, pipette transformation sample up/down vigourously with a P1000 set 500uL.
#Quick vortex
# Plate 100 uL of transformations diluted to 10^-5 on BHI plates
#Place the tubes in 37˚ hot block (will warm up tubes faster) and incubate for 90 minutes.
## do 1:10 dilutions in PBS. Before Diluting, pipette transformation sample up/down vigourously with a P1000 set 500uL.
#After incubation, spin them down again at 5,000 g/min for 1 minute and pour off supernatant into virkon
# Allow growth in a 37˚C 5% CO2 incubator for about ~48 hours.
#Resuspend cells in 300uL of pre-warmed BHI and return to 37 degree hot block for another 90 minute incubation
##colonies will be visible after 24 hours, but much easier to count after 48 hours.
#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.  
# Count colonies and calculate transformation rates
#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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