IGEM:IMPERIAL/2007/Notebook/2007-8-14

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Preparation of Stock Solutions

• 1x 1 litre of 2xYT medium in Duran bottle
• 3x 1 litre of 2xYT medium in conical flask
  • Sent to autoclave
• 1x 10 ml of pre-incubation solution for preparation of cell extract
• 6x 20 ml of 1mM IPTG stock

Preparation of Cell Extract

1. Picked a BL21(DE3) colony from Tet plate
2. Innoculated in 100 ml of 2xYT + Tet medium
3. Incubated overnight at 37 °C

Cloning of Biobricks

1. Placed 5 ml of LB in a 15 ml tube
2. Added appropriate antibiotics into the tube
3. Picked a colony from the fresh overnight plate
4. Innoculated the colony in the LB media
5. Incubated for 6 hours during the day
   • 2x 6 Biobricks cloned

Cloning of Biobricks (for Midiprep)

1. Placed 50 ml of LB in a 250 ml conical flask
2. Innoculated 1 ml of culture into LB media in flask
3. Incubated overnight at 37 °C

• 2. BBa_I13422 [ptet-GFP]
• 5. BBa_R0040 [ptet]
• 7. BBa_F2620 [plux]
• 8. BBa_F1610 [luxI]
• 11. BBa_E7104 [pT7]
• 13. BBa_B0015 [stop]
• 14. BBa_R0051 [pcI]
• 16. BBa_E0240 [GFP]
• 17. BBa_I13507 [RFP]
• 18. BBa_T9002 [plux-GFP]

Pilot Preparation of Vesicles

• The suspension prepared the day before was used to produce vesicles enclosing Tris and NaCl buffer only.

• There was a mistake in following the protocol: instead of preparing the interface with 2ml of the DOPC-mineral oil suspension and then adding 100μl of emulsion to it, the emulsion was prepared according to protocol, and then 2ml of emulsion was used to create the interface.

• Results: A sample was briefly looked at under a light microscope, but no vesicles were observed.