IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Lock and Key Decoder/2010/08/12

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Pulled cells containing pLac and GFP plasmids from 37°C room.

  • Made glycerol stock of cells containing pLac plasmid.

Added 900 μL 50% glycerol to a 2mL tube.
Added 900 μL of cells to same tube.
Mixed well and flash froze solution using dry ice.

  • Mini-prepped remaining solution

Followed Promega PureYield protocol, with the following changes.
After lysing cells and adding neutralization solution, mixture was centrifuged for 6 minutes instead of 3.
After the addition of the column wash solution, the column and collection tube were centrifuged twice to remove all traces of ethanol from the column.

  • Inoculated cells containing Lock 1 and Lock 3 because we ran out of plasmids. Will mini-prep tomorrow.
  • Began digestion/ligation/transformation of pLac promoter and Key 1 into a chloramphenicol backbone.
    • Mistakenly pulled Lock 1 plasmids from freezer. Tube was empty, and didn't realize my mistake until it was too late to perform the digestion/ligation/transformation. Will finish tomorrow.
  • Re-transformed sub-system 2a (pTet promoter, Lock 1, and GFP) using different protocol.

2 μL plasmids in 2 mL tube.
50 μL heat shock competent cells same tube.
vortexed mixture
placed mixture on ice for 10 mins.
heat shocked at 42°C for 45 seconds
placed mixture on ice for 2 mins.
Added 1000 mL SOC growth media
Placed in 37°C room for 1 hour
350 μL Plated onto Tetracycline growth media and left in 37°C room overnight