IGEM:IMPERIAL/2007/Experimental Design/Phase1/Protocol 1.1: Difference between revisions

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==Protocol==
==Protocol==
* Status - <font>Completed</font>
===Day 1===
===Day 1===
====Equipment====
====Equipment====
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*Ampicillin stock (50 mg/ml)
*Ampicillin stock (50 mg/ml)
*GFP Standard Solution <font color=red>(what is the stock concentration ?)</font>
*GFP Standard Solution - This concentration was unknown, however because we only wanted it as a positive control to show there was fluorescence and that the flurometer could measure it
<br>
<br>


'''Innoculation of Media'''
'''Innoculation of Media'''
#Inoculate 10ul of stored parts in individual 2ml LB medium containing 2ul of ampicillin <font color=red>('stored parts' means glycerol stock ? or something else ?)</font>
#Inoculate 10ul of transformed E.coli cells in individual 2ml LB medium containing 2ul of ampicillin
#Incubate at 30°C for overnight in a shaker. '''(This is to get a good stock of cells for use in the experiment. After the overnight culture the cells will be in stationary phase)''' <font color=red>(It is a funny temperature, why not 37C ?)</font>
#Incubate at 37°C for overnight in a shaker. '''(This is to get a good stock of cells for use in the experiment. After the overnight culture the cells will be in stationary phase)'''  
<br>
 
 
'''Preparation of diluted GFP standard solution'''<br>
#Add 995ul of ultra pure water an eppendorf tube, together with 5ul of undiluted GFP standard solution and mix. '''(This gives a 200x dilution to be used as a positive control)''' <font color=red>(Once prepared, where are you supposed to keep it ? -20C, -3C, room temperature ?)</font>
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<br>
<br>


===Day 2===
===Day 2===
====Equipment====
====Equipment====
*Water bath @ 30°C
*Spectrophotometer
*Stripettes
*Well-plate x1
*Well-plate x1
*Plate Centrifuge
*Fluorometer
*Stop watch
*Gilson pipettes 1000 and 200
<font color=red>(Tubes ? volume ? quantity ?)</font>
*Eppendorf tubes
 
====Reagents====
*ddH2O
*GFP standard solution
<br>
<br>


====Reagents====
'''Preparation of diluted GFP standard solution'''<br>
*LB medium
#Add 995ul of ultra pure water an eppendorf tube, together with 5ul of undiluted GFP standard solution and mix. '''(This gives a 200x dilution to be used as a positive control)'''
*Ampicillin stock (50 mg/ml)
#Place the tube on ice till it is ready to be used.
*Diluted GFP standard solution
<br>
<br>
<br>


'''Loading Plate'''
'''Loading Plate'''
#Transfer 200 µl aliquots of each of the cultures to a flat-bottomed 96 well plate. '''(Follow the schematic as shown)'''
#Transfer 200 µl aliquots of each of the cultures to a flat-bottomed 96 well plate. '''(Follow the schematic as shown)'''
: Three wells to be filled with 200µl of media to measure the absorbance background.
#Three wells to be filled with 200µl of media to measure the absorbance background.
: Three further wells to be filled with 200 µl of (media + empty-vector culture) to measure fluorescent background. (IS THIS POSSIBLE??!?!) <font color=red>(Which empty vector ? it should have the same antibiotic and the cells should also come from an overnight culture)</font>
#Standard GFP solution added as a positive control.
<br>
: Standard GFP solution added as a positive control.
 
#Place the top on the plate and centrifuge for 1 minute <font color=red>(Centrifuge ? or shacker ? if centrifuge, at which speed ?)</font>
<br>
#Remove lid and measure in the flourometer.  
#Remove lid and measure in the flourometer.  
: (Fluorescence measurements - 488 nm excitation filter, 525 nm emission filter, 0.5 seconds, CW lamp energy 12901 units)
: (Fluorescence measurements - 488 nm excitation filter, 525 nm emission filter, 0.5 seconds, CW lamp energy 12901 units)
#Repeat the measurement a further two times straight after each other '''(This is to test the variability of the machine)'''
#Repeat the measurement a further two times straight after each other '''(This is to test the variability of the machine)'''
#Place lid back on and place back in the incubator at 30<sup>o</sup>C
<font color=red>(Are you not tracking the growth of the culture too with the absorbance ? How can you normalise your fluorescence then ?)</font>
<font color=red>(Are you continuously sampling from the 10ml culture ? or are you re-using the cells in the 96-well plate ?)</font>
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'''Schematic'''
'''Schematic'''
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[[Image:icgems_invivo-testing.png|450px|top|In vivo Testing 96 well plate]]
[[Image:Icgems_invivo-testing_thur.png|450px|top|In vivo Testing 96 well plate]]
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|}
<br>
<br>
'''Collecting Data'''
#After 5 minutes of incubation measure the fluorescence by repeating procedure 3-5 above. <font color=red>(By doing so many measurements, aren't you worried to disrupt the growth of the culture with an ever changing temperature ?)</font>
<br>
#Repeat measurements after every 5 minutes until the fluorescence is constant <font color=red>(Cells are growing, so the fluorescence of the culture will not be constant before they reach stationary? Are you looking for single cell fluorescence steady-state ?)</font>
<font color=red>(You mentioned that you use only 1 96-well plate ? How do you deal with multiple measurements ?)</font>
<br>
<br>
#Before every measurement in the fluorometer spin the plates in a plate centrifuge
#Save data file from computer
#Copy and paste the data into an Excel spreadsheet for data analysis

Latest revision as of 02:25, 3 September 2007


Protocol

  • Status - Completed

Day 1

Equipment

  • 7ml sterile tubes x4
  • 1.5ml Eppendorf tube x1
  • Room temperature 25oC
  • Gilson Pipettes

Reagents

  • E.coli BL21; culture containing parts :pTet-GFP, pT7-GFP, pcI-GFP
  • LB medium
  • Ampicillin stock (50 mg/ml)
  • GFP Standard Solution - This concentration was unknown, however because we only wanted it as a positive control to show there was fluorescence and that the flurometer could measure it


Innoculation of Media

  1. Inoculate 10ul of transformed E.coli cells in individual 2ml LB medium containing 2ul of ampicillin
  2. Incubate at 37°C for overnight in a shaker. (This is to get a good stock of cells for use in the experiment. After the overnight culture the cells will be in stationary phase)


Day 2

Equipment

  • Well-plate x1
  • Fluorometer
  • Gilson pipettes 1000 and 200
  • Eppendorf tubes

Reagents

  • ddH2O
  • GFP standard solution


Preparation of diluted GFP standard solution

  1. Add 995ul of ultra pure water an eppendorf tube, together with 5ul of undiluted GFP standard solution and mix. (This gives a 200x dilution to be used as a positive control)
  2. Place the tube on ice till it is ready to be used.




Loading Plate

  1. Transfer 200 µl aliquots of each of the cultures to a flat-bottomed 96 well plate. (Follow the schematic as shown)
  2. Three wells to be filled with 200µl of media to measure the absorbance background.
  3. Standard GFP solution added as a positive control.
  4. Remove lid and measure in the flourometer.
(Fluorescence measurements - 488 nm excitation filter, 525 nm emission filter, 0.5 seconds, CW lamp energy 12901 units)
  1. Repeat the measurement a further two times straight after each other (This is to test the variability of the machine)




Schematic


Well Test Construct Stock Volume (ul) AHL (ul) Final [AHL]
A1 pTet-GFP 200 0 0
A2 pTet-GFP 200 0 0
A3 pTet-GFP 200 0 0
A5 LB-Amp Media 200 0 0
A6 LB-Amp Media 200 0 0
A7 LB-Amp Media 200 0 0
B1 pT7-GFP 200 0 0
B2 pT7-GFP 200 0 0
B3 pT7-GFP 200 0 0
B5 LB-Amp Media + Non-expressing culture 200 0 0
B6 LB-Amp Media + Non-expressing culture 200 0 0
B7 LB-Amp Media + Non-expressing culture 200 0 0
C1 pcI-GFP 200 0 0
C2 pcI-GFP 200 0 0
C3 pcI-GFP 200 0 0
C5 Diluted GFP Solution 200 0 0
C6 Diluted GFP Solution 200 0 0
C7 Diluted GFP Solution 200 2 0
D1 pTet-LuxR-pLux-GFP + 10μM AHL 200 2 10-7M
D2 pTet-LuxR-pLux-GFP + 10μM AHL 200 2 10-7M
D3 pTet-LuxR-pLux-GFP + 10μM AHL 200 2 10-7M
E1 pTet-LuxR-pLux-GFP + 100μM AHL 200 2 10-6M
E2 pTet-LuxR-pLux-GFP + 100μM AHL 200 2 10-6M
E3 pTet-LuxR-pLux-GFP + 100μM AHL 200 2 10-6M

In vivo Testing 96 well plate


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