Streptococcus mutans Transformation: Difference between revisions

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(Replaced content with "Day 1: Streaking Out Plates # Warm 1 BHI Plate # Triple streak out S. mutans from freezer Day 2: Overnight Growth Day 3: Transformations Day 4: Plating Transformations")
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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''
# Triple streak out S. mutans from freezer
##Allow this culture to grow overnight in the 37˚C 5% CO2 incubator. The other BHI culture is for a future blank.




Day 2: Overnight Growth


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


Day 3: Transformations


 
Day 4: Plating Transformations
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.

Revision as of 14:25, 15 May 2024

Day 1: Streaking Out Plates

  1. Warm 1 BHI Plate
  2. Triple streak out S. mutans from freezer


Day 2: Overnight Growth


Day 3: Transformations

Day 4: Plating Transformations

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