IGEM:IMPERIAL/2008/Prototype/Wetlab/Growth: Difference between revisions

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===Reagents===
===Reagents===
* No antibiotic should be present in the media or plates unless the strain used has a resistance or a resistance gene has previously been integrated into the strain


LB Medium
LB Medium


Lots of LB Agar plates
LB Agar plates (approximately 18 per repeat)
<font color=green> James - Would be good to know how many plates we need so we can pre make them </font>


===Protocol===
===Protocol===


'''Be VERY careful to avoid contamination at all stages'''
'''Be VERY careful to avoid contamination at all stages'''
2 repeats should also be set up and the final data averaged or used together to obtain more acurate results
A trial run should be carried out to determine the approximate dilutions required for each time point to reduce the number of plates required


#Streak a culture of ''B.subtilis'' and allow to grow overnight
#Streak a culture of ''B.subtilis'' and allow to grow overnight
#Prepare 50mL of LB medium in a flask (ideally a 500mL or 1L flask), adding the required antibiotics (if any) and take 1mL to use as a blank
#Prepare 50mL of LB medium in a flask (ideally a 500mL or 1L flask), adding the required antibiotics (if any) and take 1mL to use as a blank
#Pipette 500μL into the 50mL of LB medium and mix thoroughly
#Pipette 500μL into the 50mL of LB medium and mix thoroughly
#Immediately take 1mL of the new culture and measure the OD against the blank
#Immediately take 1mL of the new culture and measure the OD<sub>600</sub> against the blank
##Dilute 100μL of culture in 900μL of LB and mix well
##If the OD<sub>600</sub> is above 0.8, dilute 10 fold and multiply the resulting absorbance by 10
##Plate 100μL of this diluted solution
#Carry out 10 fold dilutions (100μl of culture in 900μl of LB) until the culture should be approximately dilute enough to form individual colonies (this should be determined by a trial run)
##Repeat the dilution and plating 8 times to obtain plates for colony counting
##Plate cells at the optimal dilution as well as a 10 fold higher and 10 fold lower dilution (therefore 3 diltuions and thus 3 plates will be used per time point)
###Be sure to mark the OD, time and dilution for each plate!
##Be sure to mark the OD, time and dilution for each plate!
#Grow the ''B.subtilis'' at 37°C and 225rpm of mixing
#Grow the ''B.subtilis'' at 37°C and 225rpm of mixing
#At 60 minutes, 90 minutes, 120 minutes, 150 minutes and 180 minutes take further 1 mL samples, measure OD, dilute and plate
#At 1 hour, 2 hours, 4 hours, 8 hours and overnight (~16 hours) take further 1 mL samples, measure OD, dilute and plate
<font color=green> James - We might want to capture the whole growth curve, i.e. stationary phase? So we might want to use longer sampling time. To help with this we could do a preliminary run of a dilution plate that might help us narrow down the times needed and also help in deciding which dilution will be useful so we can avoid using so many plates.</font>
<font color=red> Deffinately, particularly on the plate numbers. I'm a tad concerned about going into stationary phase as this may trigger sporulation (though that in itself may provide some interesting data)</font>
#The following day, check all plates to determine which plate at each time point is the most crowded plate on which all the colonies can still be counted individually.
#The following day, check all plates to determine which plate at each time point is the most crowded plate on which all the colonies can still be counted individually.
##The colonies on these plates should be counted, and the number of colonies mutiplied by the dilution factor to calculate the number of cells in 1mL
#The colonies on these plates should be counted on an illuminator
<font color=green>James - If we are planning to use the illuminator for colony counting it might be useful to have a guide on how to set it up? Also we should test how accurate it is at counting as might slight errors in dilution plating get amplified </font>
##To use the illuminator...
 
#The number of colonies on the plates should be multiplied by the dilution to obtain the colony forming units at each time point
<font color=red> I've never used an illuminator so if you could add a bit about it that would be grand. And yes, we should test for accuracy on a trial run</font>
#Plot a calibration curve of OD<sub>600</sub> against cells per mL or OD<sub>600</sub> against colony forming units
#Plot a calibration curve of OD<sub>600</sub> against cells per mL or OD<sub>600</sub> against colony forming units

Revision as of 10:21, 25 August 2008

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<html><a href=http://openwetware.org/wiki/IGEM:IMPERIAL/2008/Prototype><img width=50px src=http://openwetware.org/images/f/f2/Imperial_2008_Logo.png></img</a></html> Home The Project B.subtilis Chassis Wet Lab Dry Lab Notebook

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Cell Count v Optical Density Curve Calibration

Aim

To produce a calibration curve to aid in the normalising of fluorescence values to allow proper characterisation of Promoters and RBSs for B.subitlis. This protocol must give results that are as accurate as possible over a considerable range of Optical Densities.

Equipment

Spectrophotometer

Cuvettes

P20, P200 and P1000 Gilsons

50mL flask (an additional one for each repeat)

Timer

Reagents

  • No antibiotic should be present in the media or plates unless the strain used has a resistance or a resistance gene has previously been integrated into the strain

LB Medium

LB Agar plates (approximately 18 per repeat)

Protocol

Be VERY careful to avoid contamination at all stages

2 repeats should also be set up and the final data averaged or used together to obtain more acurate results

A trial run should be carried out to determine the approximate dilutions required for each time point to reduce the number of plates required

  1. Streak a culture of B.subtilis and allow to grow overnight
  2. Prepare 50mL of LB medium in a flask (ideally a 500mL or 1L flask), adding the required antibiotics (if any) and take 1mL to use as a blank
  3. Pipette 500μL into the 50mL of LB medium and mix thoroughly
  4. Immediately take 1mL of the new culture and measure the OD600 against the blank
    1. If the OD600 is above 0.8, dilute 10 fold and multiply the resulting absorbance by 10
  5. Carry out 10 fold dilutions (100μl of culture in 900μl of LB) until the culture should be approximately dilute enough to form individual colonies (this should be determined by a trial run)
    1. Plate cells at the optimal dilution as well as a 10 fold higher and 10 fold lower dilution (therefore 3 diltuions and thus 3 plates will be used per time point)
    2. Be sure to mark the OD, time and dilution for each plate!
  6. Grow the B.subtilis at 37°C and 225rpm of mixing
  7. At 1 hour, 2 hours, 4 hours, 8 hours and overnight (~16 hours) take further 1 mL samples, measure OD, dilute and plate
  8. The following day, check all plates to determine which plate at each time point is the most crowded plate on which all the colonies can still be counted individually.
  9. The colonies on these plates should be counted on an illuminator
    1. To use the illuminator...
  10. The number of colonies on the plates should be multiplied by the dilution to obtain the colony forming units at each time point
  11. Plot a calibration curve of OD600 against cells per mL or OD600 against colony forming units
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