Procedure #1 – Isotope Efficient Protocol (New)

  1. Transformed cells are grown in 1L of LB at 37C shaken at 250 rpm.
  2. The LB culture is grown until it reaches an optical density of ~0.7 at 600nm (~5 hours).
  3. The culture is gently pelleted by spinning at 5000 x g for 30 minutes.
  4. 4 – 1L cell culture pellets are resuspended in 1L of M9 minimal salts media (resulting in an OD = 2.8).
  5. The resuspended cell pellets are then shaken at 250 rpm at 37C for 1 hour.
  6. Protein expression is induced by the addition of 0.8 mM IPTG.
  7. The M9 cultures are allowed to grow for at least 4 hours before the cells are harvested.

Reference: Journal of Biomolecular NMR, 20: 71-75, 2001.

Procedure #2 – Growth Protocol

The principal consideration to take into account when growing the cells is to minimize the total culture time prior to induction. This is especially important with cells transformed with AMP resistant plasmids as the selection will be lost during the course of the growth owing to the fact that b-lactamase (which degrades ampicillin) is secreted into the medium. The way that we typically perform growths on minimal medium is to perform a fresh transformation of the plasmid of interest into Novagen BL21(DE3) cells onto a LB+antibiotic plate at about 5 PM on the night before the growth. The next morning, the components that comprise the media are mixed (as indicated above).

The culuture is inoculated simply by picking approximately 10 colonies or so from the plate (with a sterile toothpick) into each liter of the complete M9 medium. When inoculated in this manner, the Novagen BL21(DE3) cells will typically rise to an A600 of about 0.7 in a period of about 7 hours. IMPORTANT NOTE: At the point of induction of protein expression, 2.5 mL of sterile 20% (w/v) D-glucose is added.