Streptococcus mutans Transformation: Difference between revisions

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Day 1:
Day 1:
#Label and add 3ml of BHI to two sterile test tubes
##Label and add 3ml of BHI to two sterile test tubes
#Using a green inoculation loop, transfer 3 colonies of S. mutans
##Using a green inoculation loop, transfer 3 colonies of ''S. mutans''
#Allow this culture to grow overnight in the 37˚C 5% CO2 incubator. The other BHI culture is for a future blank.
##Allow this culture to grow overnight in the 37˚C 5% CO2 incubator. The other BHI culture is for a future blank.
 




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##Get a sterile test tubes for each strain, and one for the blank.
##Get a sterile test tubes for each strain, and one for the blank.
##Fill the tubes
##Fill the tubes
*Strains to transform: 3 ml of BHI
##*Strains to transform: 3 ml of BHI
*Blank: 5-7ml 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.  
##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
##Place a mini-vortexer by the flame
##Prepare 500ml Virkon  
##Prepare 500ml Virkon  


Inoculation:
Inoculation:
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##Place these into the 37˚C 5% CO2 incubator. Allow them to grow for 2 hours and 30  minutes before checking them.
##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.
##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:
Washing the cells — BSL2 hoods:
##Label enough microtubules for conditions and replicates
##Label enough microtubules for conditions and replicates
##First wash:
##First wash:
##Add 300 uL of the culture to these tubes
##*Add 300 uL of the culture to these tubes
##Spin them down for 2 minute at 8,000 grams
##*Spin them down for 2 minute at 8,000g
##Pipette out 300uL of the supernatant into Virkon
##*Pipette out 300uL of the supernatant into Virkon
##Resuspend in 300uL of appropriate media
##*Resuspend in 300uL of appropriate media
##Second Wash
##Second Wash
##Spin them down for 1 minute at 5,000 grams
##*Spin them down for 1 minute at 8,000g
##Dump out supernatant into Virkon
##*Dump out supernatant into Virkon
##Resuspend in 300uL of appropriate media
##*Resuspend in 300uL of appropriate media
##Respuspension
##Respuspension
##Spin them down for 1 minute at 5,000 grams
##*Spin them down for 1 minute at 8,000g
##Dump out supernatant into Virkon
##*Dump out supernatant into Virkon
##Add in 300uL of appropriate media
##*Add in 300uL of appropriate media
 
 


Adding XIP and DNA:
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.  
##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.
##Set aside negative control tubes, nothing more will be added to them
##Place all of the tubes in the 37˚C Dry heat-block for 2.5 hours.
##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.
##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)
##Label dilution tubes for appropriate strains and concentrations (for both transformation dilutions and dilutions of total cells)
##Total cells: 10-5
##*Total cells: 10-5
##Transformation dilutions: 10-1  
##*Transformation dilutions: 10-1  
##Allow all plates to grow for 36-48 hours in the 37˚C 5% CO2 incubator before counting colonies.
##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.

Revision as of 15:11, 27 July 2023

Day 1:

    1. Label and add 3ml of BHI to two sterile test tubes
    2. Using a green inoculation loop, transfer 3 colonies of S. mutans
    3. Allow this culture to grow overnight in the 37˚C 5% CO2 incubator. The other BHI culture is for a future blank.


Day 2: Setting up

    1. Turn on the spectrophotometer
    2. Turn on the dry water bath, fill 8 of the divots with DI water and set to 37˚C.
    3. Get a sterile test tubes for each strain, and one for the blank.
    4. Fill the tubes
      • Strains to transform: 3 ml of BHI
      • Blank: 5-7ml of BHI
    5. Warm all of the tubes in the large divots in the 37˚C heat block; allow these to warm for at least 20 minutes.
    6. Place a mini-vortexer by the flame
    7. Prepare 500ml Virkon


Inoculation:

    1. After 20 minutes of warming, retrieve tubes.
    2. Grab overnight samples, vortex them and add 150uL (1:20 dilution) of the appropriate culture to the new tubes.
    3. Place these into the 37˚C 5% CO2 incubator. Allow them to grow for 2 hours and 30 minutes before checking them.
    4. 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:

    1. Label enough microtubules for conditions and replicates
    2. 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
    3. Second Wash
      • Spin them down for 1 minute at 8,000g
      • Dump out supernatant into Virkon
      • Resuspend in 300uL of appropriate media
    4. 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:

    1. To achieve a final concentration of 2uM of XIP, add 6 uL of 100uM XIP will be added to each tube.
    2. Set aside negative control tubes, nothing more will be added to them
    3. Add 300ng of DNA to each tube (with the exception of the negative controls) and then vortex.
    4. Place all of the tubes in the 37˚C dry heat-block for 2.5 hours.
    5. Place appropriate number of 1000ug/mL Spectinomycin plates in the incubator, and appropriate number of BHI plates in the incubator.
    6. 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
    7. Allow all plates to grow for 36-48 hours in the 37˚C 5% CO2 incubator before counting colonies.
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