User:Stefan J. Tekel/Notebook/Pc-TF Engineering/2015/11/17

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Full Length PcTF Expression in Rosetta 2p Lys Cells

Objective: Determine if PcTF will express in bacteria

1. Innoculate 25 mL LB/Kan with overnight culture to final OD600 = 0.1
2. Grow to OD600=1.0 at 37°C
3. Induce protein expression with 25 μL of 1.0 M IPTG to a final concentration of 1.0 mM
4. Grow at 37°C for 2 hours

Take 750 μL of an uninduced and induced sample for gel analysis.
Spin cells at max speed for one minute and decant supernantant. Resuspend cells in 60 μL of water, and add 15 μL 4x protein loading dye. Heat at 90 °C for 5 minutes. Transfer to a 0.22 micron spin filter and spin at max speed for 2 minutes. This will remove cell debris and allow the protein to be easily loaded onto a gel.

4-12% PAGE (200 V for 35 minutes in 1x MOPS buffer)
Lane 1 and 6 is the ladder, Lane 2 is GFP uninduced, lane 3 is induced GFP. Lane 4 is PcTF uninduced, and lane 5 is induced PcTF