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		+	= Wet Lab: Protocols: Initial Testing of DNA Constructs ''in vivo''  =
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	'''Aims'''		'''Aims'''
	*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.

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Revision as of 04:27, 22 October 2007

• Home
  • Site Map
  • Meet the Team
  • Fun with iGEM
  • IcGEMs@OpenWetware
  • Acknowledgements
• Infector Detector
  • Introduction
  • Specification
  • Design
  • Modelling
  • Implementation
  • Testing/Validation
  • F2620 Comparison
  • Conclusion
• Cell by Date
  • Introduction
  • Specification
  • Design
  • Modelling
  • Implementation
  • Testing/Validation
  • Conclusion
• Cell-Free
  • What is Cell-Free?
  • Cell-Free Vs. Cell
  • Our Contributions
  • Characterisation
  • Applications
• Wet Lab
  • Lab Notebook
  • Protocols & Results
  • DNA Constructs
• Dry Lab
  • Modelling
  • Data Analysis
  • Software

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