Imperial/Wet Lab/Protocols/CBD1.1

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__NOTOC__
__NOTOC__
= Wet Lab: Protocols: Initial Testing of DNA Constructs ''in vivo''  =
= Wet Lab: Protocols: Initial Testing of DNA Constructs ''in vivo''  =
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==Aims==
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'''Aims'''
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*To test to see if the DNA constructs from the registry are viable. This is done in vivo.
*To test to see if the DNA constructs from the registry are viable. This is done in vivo.
*The constructs are pTet-GFP and pT7-GFP.
*The constructs are pTet-GFP and pT7-GFP.
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===Day 1===
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====Equipment====
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==Day 1==
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===Equipment===
*7ml sterile tubes x4
*7ml sterile tubes x4
*1.5ml Eppendorf tube x1
*1.5ml Eppendorf tube x1
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*Gilson Pipettes p1000 p200 p20
*Gilson Pipettes p1000 p200 p20
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====Reagents====
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===Reagents===
*''E.coli'' BL21; culture containing parts :pTet-GFP, pT7-GFP, pcI-GFP
*''E.coli'' BL21; culture containing parts :pTet-GFP, pT7-GFP, pcI-GFP
*LB medium
*LB medium
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<br>
<br>
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====Protocol====
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===Protocol===
'''Innoculation of Media'''
'''Innoculation of Media'''
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#Inoculate 10ul of transformed E.coli cells in individual 2ml LB medium containing 2ul of ampicillin
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#Inoculate 10ul of transformed E.coli cells in individual 2ml LB medium containing 2ul of ampicillin.
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#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)'''  
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#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>
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===Day 2===
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==Day 2==
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====Equipment====
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===Equipment===
*1 Fluorometer plate (black)
*1 Fluorometer plate (black)
*Fluorometer + PC
*Fluorometer + PC
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*Eppendorf tubes
*Eppendorf tubes
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====Reagents====
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===Reagents===
*LB medium
*LB medium
*E.coli culture with transformed plasmid  
*E.coli culture with transformed plasmid  
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<br>
<br>
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====Protocol====
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===Protocol===
'''Preparation of diluted GFP standard solution'''<br>
'''Preparation of diluted GFP standard solution'''<br>
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#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)'''  
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#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.
#Place the tube on ice till it is ready to be used.
<br>
<br>
<br>
<br>
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====Loading Plate====
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'''Loading Plate'''
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#Transfer 200 µl aliquots of each of the cultures to a flat-bottomed 96 well plate. '''(Follow the schematic as shown.)'''
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#Transfer 200 µl aliquots of each of the cultures to a flat-bottomed 96 well plate. '''(Follow the schematic as shown)'''
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#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.
#Standard GFP solution added as a positive control.
#Standard GFP solution added as a positive control.

Latest revision as of 02:21, 27 October 2007



Wet Lab: Protocols: Initial Testing of DNA Constructs in vivo

Aims

  • To test to see if the DNA constructs from the registry are viable. This is done in vivo.
  • The constructs are pTet-GFP and pT7-GFP.

Day 1

Equipment

  • 7ml sterile tubes x4
  • 1.5ml Eppendorf tube x1
  • 37°C incubator
  • Gilson Pipettes p1000 p200 p20

Reagents

  • E.coli BL21; culture containing parts :pTet-GFP, pT7-GFP, pcI-GFP
  • LB medium
  • Ampicillin stock (50 mg/ml)


Protocol

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

  • 1 Fluorometer plate (black)
  • Fluorometer + PC
  • Gilson pipettes 1000 and 200
  • Eppendorf tubes

Reagents

  • LB medium
  • E.coli culture with transformed plasmid
  • GFP standard solution
  • ddH2O


Protocol

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
C5 Diluted GFP Solution 200 0 0
C6 Diluted GFP Solution 200 0 0
C7 Diluted GFP Solution 200

In vivo Testing 96 well plate