BL21(DE3) E. coli strain, competent cells

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Description

BL21(DE3) E. coli are ideal foruse with bacteriophage T7 promoter-based expression systems (e.g.,pRSET, pCR¤T7, and pET). BL21(DE3) carry the lambda DE3 lysogen. Recombinantproteins that are non-toxic toE. coliare generallyexpressed at higher levels in BL21(DE3) cells than in BL21(DE3)pLysS orBL21(DE3)pLysE. However, the basal expression levels of heterologous genes aresignificantly higher in BL21(DE3) than in BL21(DE3)pLysS or BL21(DE3)pLysE.

Genotype

F – ompT hsdSB (rB– mB–) gal dcm(DE3)

Recovery

1.Obtainan LB agar plate with the appropriate antibiotic.

2.Usinga sterile pipette tip, touch the bacteria growing within the punctured area ofthe stab culture. (A sterilized wire loop or sterile toothpick can be used inplace of a sterile pipette tip.)

3.Runthis tip lightly over a section of the plate, as shown in the figure, to createstreak #1.

4.Usinganother sterile pipette tip, pass through streak #1 and spread the bacteriaover a second section of the plate, to create streak #2.

5.Usinga third sterile pipette tip, pass through streak #2 and spread the bacteriaover the last section of the plate, to create streak #3.

6.Growovernight in a 37 ^o C incubator (unless a different growthtemperature is indicated on the plasmid datasheet).

7.Inthe morning, single colonies should be visible. If the bacterial growth is toodense, re-streak onto a new agar plate to obtain single colonies.