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

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8/12
Pulled cells containing pLac and GFP plasmids from 37°C room. Procedure: 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.
 * Made glycerol stock of cells containing pLac plasmid.


 * Mini-prepped remaining solution

Procedure: 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.

Procedure: 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
 * Matthew Entler 18:08, 12 August 2010 (EDT):


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