My first post here - hello!
I've got given the following protocol for making competent cells (see below). It has been established in a lab in France for a long time, but even intensive searching has only thrown up some limited information about it.
What strikes me particularly odd is the treatment buffer ('FB' I assume stands for freezing buffer). It's not much of a buffer with KAc at a pH at 7.5 and then set to 6.2, as it's far off acetate's pKa.
I know historically making competent cells was an arcane art and researchers like Hanahan would only give out cell aliquots and not divulge their painstakingly developed procedures.
Is this 'buffer' one of these odd quirks? Has anyone come across this before, maybe even this protocol?
PREPARATION OF COMPETENT CELLS (FB METHOD) • set up an overnight culture of TOP10F' or other E. coli strain in SOB (or LB) and let grow O/N with shaking • inoculate 100 ml of SOB with 1 ml of the saturated O/N culture • grow until OD550 = 0.45 to 0.55 (getting the OD right is important as it affects the transformation efficiency**) • transfer culture to 2-50 ml Falcon tubes and chill on ice for 10' • centrifuge 10' at 2500 rpm at 4°C • resuspend each 50 ml cell pellet in 15 ml FB buffer • let sit on ice for 10' • centrifuge 10' at 5000 rpm at 4°C • resuspend each pellet in 4 ml FB buffer with gentle pipetting • aliquot cells, freeze in liquid nitrogen and store at -80°C ______________________________________________________________________ SOB 2% tryptone 0.5% yeast extract 10 mM NaCl 2.5 mM KCl 10 mM MgCl2** 10 mM MgSO4** ** add after autoclaving FB 100 mM KCl 50 mM CaCl2 10% glycerol 10 mM KAc pH 7.5 adjust the pH of the solution to 6.2 with the addition of HCl autoclave Getting the OD right……. Ln[Y/start]/K = X Where Y = population size (cell number, OD etc.) X = time (typically in minutes) K = slope = rate constant; doubling time = 0.693/K Have determined K experimentally twice for TOP10F’ (K=0.0183 – single determination; K=0.01974 – growth curve performed on 02 May 06)