IGEM:IMPERIAL/2007/Wet Lab/Protocols/CBD1.1

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(Reagents)
(Reagents)
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====Reagents====
====Reagents====
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*ddH2O
+
*LB medium
 +
*E.coli culture with transformed plasmid
*GFP standard solution
*GFP standard solution
 +
*ddH2O
<br>
<br>

Revision as of 12:28, 20 October 2007

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.

Contents

Day 1

Equipment

  • 7ml sterile tubes x4
  • 1.5ml Eppendorf tube x1
  • 37°C incubator
  • Gilson Pipettes 1000 200 20 10

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


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