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==== | ||
*7ml sterile tubes x4 | *7ml sterile tubes x4 | ||
*1.5ml Eppendorf tube x1 | *1.5ml Eppendorf tube x1 | ||
* | *Room temperature 25<sup>o</sup>C | ||
*Gilson Pipettes | *Gilson Pipettes | ||
====Reagents==== | ====Reagents==== | ||
*''E.coli'' BL21; culture containing parts | *''E.coli'' BL21; culture containing parts :pTet-GFP, pT7-GFP, pcI-GFP | ||
*LB medium | *LB medium | ||
*Ampicillin stock (50 mg/ml) | *Ampicillin stock (50 mg/ml) | ||
*GFP Standard Solution | *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 | #Inoculate 10ul of transformed E.coli cells in individual 2ml LB medium containing 2ul of ampicillin | ||
#Incubate at | #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> | <br> | ||
===Day 2=== | ===Day 2=== | ||
====Equipment==== | ====Equipment==== | ||
*Well-plate x1 | *Well-plate x1 | ||
* | *Fluorometer | ||
* | *Gilson pipettes 1000 and 200 | ||
*Eppendorf tubes | |||
====Reagents==== | ====Reagents==== | ||
* | *ddH2O | ||
* | *GFP standard solution | ||
<br> | <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)''' | ||
#Place the tube on ice till it is ready to be used. | |||
<br> | <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. | |||
#Standard GFP solution added as a positive control. | |||
#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)''' | ||
<br> | |||
<br> | |||
<br> | <br> | ||
'''Schematic''' | '''Schematic''' | ||
{| border="1" cellpadding="1" | {| border="1" cellpadding="1" | ||
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[[Image: | [[Image:Icgems_invivo-testing_thur.png|450px|top|In vivo Testing 96 well plate]] | ||
|} | |} | ||
<br> | <br> | ||
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
- Inoculate 10ul of transformed E.coli cells in individual 2ml LB medium containing 2ul of ampicillin
- 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
- 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)
- Place the tube on ice till it is ready to be used.
Loading Plate
- 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.
- Standard GFP solution added as a positive control.
- 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)
- Repeat the measurement a further two times straight after each other (This is to test the variability of the machine)
Schematic
|