What steps can I take to increase the transformation efficiency of electroporated Cg? When transforming with a plasmid from E. coli, the efficiency is very high and I have ample colonies to choose from, but I'd like to skip the E. coli step and transform the ligation reaction mix directly in to Cg. Unfortunately, that has been unsuccessful/low colony numbers. Any suggestions are greatly appreciated.
You could try weakening the cell wall of the bacterium to increase cytoplasmic membrane fluidity. See the linkArticle Improving the electro-transformation efficiency of Corynebac...
Article High efficiency electroporation of intact Corynebacterium gl...
Alternatively, you could try the method for Corynebacterium pseudotuberculosis. Grow a single colony of the bacterium overnight (18 h). Dilute in fresh medium (1:50) and then incubate for 72 h to an optical density at 600 nm (OD 600 nm) = 1.0– 1.5. You could add 1.5% glycine (v/v) to the culture at OD 600 nm = 0.5, with growth till OD 600 nm = 1.0–1.5. Harvest cells by centrifugation at 5000 rpm at 4 deg C for 20 min. Wash cell pellets four times with 40 mL of ice-cold 10% glycerol in water (v/v). Following the last centrifugation, re-suspend cells in 1 ml 10% glycerol. Finally, freeze 100 mL aliquots in dry ice and methanol and store at -70 deg C. Read the attached for details as well as the electro-transformation assay.
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