Making competent E.coli is getting me desperate - After succeeding once, all tries after keep failing (Apr/27/2006 )

i agree with aaiyada. I always streak on selective plate (if applicable regarding antibiotic) before starting a new culture.

and absolutely chill the cells on ice at every step, and add chilled milliQ and glycerol and everything you use...and thaw them on ice before transforming...you're peppering their little membranes with holes and making them very sensitive to everything

(btw, I think they don't really have holes..the membrane's just weaker...but if you visualize it that way it helps you be suitably careful)

Here is exactly the protocol I have used for decades without fail. In fact, I wrote that page in 1995, and it was picked up by the USDA and thus still survives on the web.

If you follow that protocol exactly (including growing the overnight and sub-culturing the next morning), I guarantee your cells will be competent, and remain so at -80°C for a considerable time (months).

I have competent e. coli. Thank you all so much for your help. I am now working detergent-free, always on ice (even pipettes, tubes,...) and fast as lightning

I don't know what did the final trick, but i assume it was a problem with the bacteria...since i took a new tube it worked.

Thankx again!