IPTG Induction from a T7 Promoter

 

  1. Prepare 1L of LB for lab use and pour 500ml of the LB into each of the two baffled flasks
  2. Autoclave the baffled flasks on the P1 cycle.  If the autoclave is in use leave the flasks with the LB inside in the cold room while you are waiting.
  3. Grab a plate containing your desired protein form the cold room
  4. Make a couple 5ml test tubes of LB and transfer a single colony into the test tube and allow the cultures to grow overnight in the 37 degree incubator.
  5. After the overnight is complete fill two 50mL conical tubes with about 20mL LB, 20uL of your drug of choice, and about 20uL of your culture and allow it to grow in the 37 degree incubator.
  6. Once your culture is ready (it should take about 2½ hours), add 500uL of the drug you are using to each of the two large baffled flasks.
  7. Take about 5mL of the culture that you grew and place it in the 500ml flasks.
  8. Put the large baffled flasks in the 37 degree incubator and let them grow until 0.50 OD (2½ hours.)
  9. Once the culture has reached 0.50 OD you need to take a 500uL sample of both of the flasks and label them correctly as the “pre” induction samples.  Spin them down at a speed of 4000 for about 5 minutes and store them in the fridge.
  10. Then add enough IPTG so that the final concentration is 1mM (If you have a 500ml culture you should add 5ml of the 100mM IPTG)
  11. Then incubate the culture at 30 degrees for 3-7 hours or overnight if necessary. 
  12. When the culture is done grab two of the large plastic bottles for the centrifuge and label them.
  13. Pour a 500ml culture into each of the two bottles.
  14. Then take another 500uL sample from both of the flasks and label them the “post” induction samples. 
  15. Spin the bottles down in the huge centrifuge in the Fambrough Lab being very careful to fully tighten the rotors.  Spin at a speed of 4,000 for about 10-12 minutes.
  16. Once the centrifuge is done bring the bottles back and empty the supernatant back into the flasks. 

17.  Place the labeled bottles into the very top section of the -80 freezer for later use.