Streptococcus mutans Transformation: Difference between revisions

From Microbial Ecology and Evolution Lab Wiki
Jump to navigation Jump to search
No edit summary
(updating protocol)
 
(3 intermediate revisions by the same user not shown)
Line 1: Line 1:
Day 1: Streaking Out Plates
Day 1: Streaking Out Plates
# Warm 1 BHI Plate
# Warm 1 BHI Plate
# Triple streak out S. mutans from freezer
# Streak out S. mutans from freezer with inoculating loop in a BSL II hood.
# Allow overnight growth in a 37˚C 5% CO2 incubator
# Allow overnight growth in a 37˚C 5% CO2 incubator.


Day 2: Overnight Growth
Day 2: Overnight Growth
# Add 1500uL of BHI to a well of the 96-well plate
# Add 1000uL of SMUR to the number of wells you will need in a 96-well plate.
# Add ~5 colonies of S. mutans to the BHI
# Add 1 colony of S. mutans to each SMUR well.
# Allow overnight growth in a 37˚C 5% CO2 incubator
# Allow overnight growth in a 37˚C 5% CO2 incubator
   
   
Day 3: Transformations
Day 3: Transformations
#Add 1500uL of SMUR to a well of the 96-well plate
DO the following steps in a BSL II hood.
## Have one well set aside for  
# 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 4: Plating Transformations
# Warm BHI plates (for total cell count) and BHI plates with appropriate selection antibiotic.
# Plate 100 uL of undiluted  negative control (sample with no DNA or XIP) on an antibiotic BHI plate.
# Plate 100 uL of transformations diluted to 10^-2 on antiobiotic BHI plates
## do 1:10 dilutions in PBS. Before Diluting, pipette transformation sample up/down vigourously with a P1000 set 500uL.
# Plate 100 uL of transformations diluted to 10^-5 on BHI plates
## do 1:10 dilutions in PBS. Before Diluting, pipette transformation sample up/down vigourously with a P1000 set 500uL.
# Allow growth in a 37˚C 5% CO2 incubator for about ~48 hours.
##colonies will be visible after 24 hours, but much easier to count after 48 hours.
# Count colonies and calculate transformation rates

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
Retrieved from "http://microbes.sites.haverford.edu/MEE_wiki/index.php?title=Streptococcus_mutans_Transformation&oldid=3443"