IGEM:IMPERIAL/2007/Notebook/2007-9-11

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'''Preparations'''<br>
'''Preparations'''<br>
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==Cloning of Biobricks==
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#Placed 5 ml of LB in a 15 ml tube
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#Added appropriate antibiotics into the tube
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#Picked 6 colony from the fresh overnight plate
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#Innoculated the colony in the LB media
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#Incubated overnight at 37 °C
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#*6x1 Biobricks cloned
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==Maxiprep of Biobricks (preparation)==
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#Cells were spun down and pelleted
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#Cells stored at -20 °C for Maxiprep tomorrow

Revision as of 08:34, 11 September 2007

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Vesicles

Formation of Vesicles

Three commercial S30 cell extract mixes were prepared, with the ratio of 1:4:3 AA Mixture : Premix : S30 Extract. These were then used to form three reactions:

  • 50μl Negative Control: 33,3μl of S30 mix, 16,7μl nuclease free water.
  • 50μl Experiment: 33,3μl of S30 mix, 3,8μl Plux GFP DNA, 12,9μl nuclease free water.
  • 20μl Positive Control: 13,4μl of S30 mix, 1,5μl Plux GFP DNA, 5,1μl nuclease free water.

Two of the three suspensions prepared the day before were used to prepare emulsions containing commercial S30 cell extract mixes:

  • Experiment: 10ml emulsion with 50μl of S30 cell extract with Plux GFP DNA
  • Negative Control: 10ml emulsion with 50μl of S30 cell extract without DNA.

The third suspension was used to prepare four interfaces (2ml suspension over 3ml Solution A), to become four samples:

  • Sample 1: Experiment, with circulation by syringe
  • Sample 2: Experiment, without circulation by syringe
  • Sample 3: Negative Control, with circulation by syringe
  • Sample 4: Negative Control, without circulation by syringe

The remaining 20μl of S30/DNA reaction was used to form postitive controls:

  • Concentrated Positive Control: 15μl of S30/DNA reaction
  • Diluted Positive Control: 5μl of S30/DNA reaction diluted into 295μl of Solution A

AHL was then added to each of the four samples and the two positive controls above, to reach a concentration of 1μM/dm3.


Results


Preparations


Cloning of Biobricks

  1. Placed 5 ml of LB in a 15 ml tube
  2. Added appropriate antibiotics into the tube
  3. Picked 6 colony from the fresh overnight plate
  4. Innoculated the colony in the LB media
  5. Incubated overnight at 37 °C
    • 6x1 Biobricks cloned

Maxiprep of Biobricks (preparation)

  1. Cells were spun down and pelleted
  2. Cells stored at -20 °C for Maxiprep tomorrow
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