BL21 (DE3) transformation

This protocol is used in heat induced transformations. It is adapted from the Promega protocol for their BL21 (DE3) cells. I also like to use Promega competent cells. These cells are located in the -80 AA2.5c.

Genotype. Promega now sells this strain only as BL21 (DE3) pLysS (L1191, 5 × 200 µl; L1195, 20 × 50 µl):

F^–, ompT, hsdS_B (r_B^–, m_B^–), dcm, gal, λ(DE3), pLysS, Cm^r

BL21 (DE3) without pLysS has the same genotype minus pLysS and Cm^r. pLysS expresses T7 lysozyme, which lowers leaky expression from T7 promoters; it is chloramphenicol resistant, so keep chloramphenicol in the plates and medium.

Contents

1. Protocol

1. Protocol

1. Thaw a tube of Promega BL21 (DE3), or other heat inducible competent cells, on ice.
2. Add 5-10 ng DNA in a volume of 10 µl cells. (Never re-freeze competent cells. Use the rest for additional transformations or throw away.)
3. Incubate the cells on ice for 30 mins.
4. Heat shock at 42’C for 30 secs.
5. Place on Ice for 2 mins.
6. Add 250 µl RT SOC to cells.
7. Place in the 37° incubator for 1 hour shaking at 225 rpm.
8. Plate 100 µl of cells on the appropriate antibiotic plate. This will be determined by the selection markers of your plasmid.