Team:Washington/Notebook/protein gel


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Protein Gel

  1. Set up overnights of parts J36848-J36851
  2. Dilute 1μL overnight into 1mL broth
  3. Add 1 mM IPTG and let grow for four hours
  4. After cells have grown up to saturation, heat water to a boil
  5. Add 100μL of overnight to a 1.5mL tube
  6. Pellet by spinning at max speed for 30 secs in the microcentrifuge
  7. Discard supernatant
  8. Pull an aliquot of 5x sample loading buffer out of the freezer and thaw
  9. Add 20uL BME to aliquot
  10. Resuspend samples in 50μL sample loading buffer (pipette up/down)
  11. Boil samples for 10 minutes
  12. While boiling, prepare 500mL 1x SDS buffer:
    1. 50mL 10x buffer to 450mL water
    2. Take a gel out of the fridge and and put it in the gel box (keep the gel container for staining!!!)
    3. Pour the mixed buffer solution into the half of the gel box that the gel is in
    4. Remove the gel comb
    5. Fill the little container on the top of the gel until it's about 0.5 cm from the top with buffer
    6. Remove any bubbles in the wells
  13. Vortex samples
  14. Spin down samples for a few seconds
  15. Load 3μL into each well
  16. Load 10μL of ladder in appropriate wells
  17. Run at 180V until the dye is about to fall off the gel