IPTG Induction for Protein Expression

Here is a scraping protocol for large scale induction and harvesting of transformed E. coli (typically BL21 or NiCo21 strains), starting with already transformed colonies. For expression of some proteins, scraping bacterial lawns from plates seems to increase the overall yield. Frozen cell pellets can be stored almost indefinitely in a -80 freezer until they are needed for lysis and protein purification.

  1. Inoculate 3 transformed colonies in 200 ul LB-amp-cam broth.

  2. Plate the 200 ul broth mixture on LB-amp-cam plates. Incubate overnight at 37 oC.

  3. You can prepare additional plates in this step if desired to decrease the time needed to reach the necessary optical density

  4. Prewarm 1 L LB amp/cam in a 2 L fernbach flask at 37 oC.

  5. Scrape culture from plate media to the broth until an OD600 of 0.1 is reached. .

  6. Grow the cells at 37 oC on a 225 RPM shaker to an OD600 of 0.5-0.8. Check the OD600 frequently using 1 ml of culture in a cuvette to prevent overgrowth.

  7. Add 10 ml 100 mM IPTG to the 1 L culture and grow for 2 hours.

  8. Spin cells down 200 ml at a time in 250 ml sorvall bottles using the Sorvall RC5 centrifuge. Spin for 5 minutes at 6500 G and 4 oC. Perform multiple spins to spin down all culture media.

  9. Freeze cell pellets at -80 C.


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