Preparation of Glycerol Stocks
The BL21 cells are fairly fussy in that, unlike other E. coli strains, their plasmids become unstable if the cells are stored in glycerol concentrations greater than 10%. For this reason, the cells should be stored in 8% glycerol and frozen quickly in liquid nitrogen.
Freezing of the BL21 cells poses a problem as this strain has been developed so that it can”self-lyse” if freeze-thawed. The pLysS plasmid [under chloramphenicol pressure] expresses T7lysozyme which, in addition to inhibiting RNA polymerase, also can lyse the E. coli cells if the cell wall integrity is compromised. Therefore, ensure that you freeze the cells as fast as possible.
- 5 ml LB broth in universal bottles. Add chloramphenicol to 34 μg/ml and either kanamycin to 25 μg/ml or the appropriate ampicillin conc. Add antibiotics just before addition of the cells.
- 80% glycerol. Decant ~10 ml glycerol from a stock bottle and microwave until hot and runny [otherwise the glycerol is too viscous]. Measure out 8 ml and add 2 ml distilled water. Mix well and keep on ice until chilled.
- Bacteria, grown overnight on LB agar, supplemented with antibiotics.
- Autoclaved Eppendorf tubes.
- Liquid nitrogen.
- Inoculate 5 ml LB broth with a single colony. Transfer to the 30°C incubator and shake overnight (~14-16 h). Make sure that the bottles are inclined while shaking, but not completely horizontal. Ensure that the lids are sealed correctly and use extra Parafilm to be sure.
- Remove the bottles from the incubator and place on ice.
- Mark the Eppendorf tubes well.
- Add 100 μl cell solution per Eppendorf and add 900 μl cold 80% glycerol. Mix gently, but thoroughly. Plunge into liquid nitrogen immediately. If there is enough space in the freezer boxes, you could split the cells into e.g., 5 x 200 μl aliquots.
- When growing up from glycerol stocks, scrape the top layer of frozen cells with a sterile yellow pipette tip [do this at the 70°C freezer – do not remove the frozen cells from the freezer]. Drop the tip into LB with appropriate antibiotics and grow overnight.