How do I make more competent cells? - by growing up and freezing glycerol stock? (Jun/26/2005 )
I have some BL21DE3 bacteria for expression. How do I make more of it? Can I just grow some overnight, dilute and let it grow until mid log phase and take some for glycerol stock? When these are thawed, will they be the same cells that can be used for transformation and expression?
I think it is ok according to what you said above,you can get more BL21DE3 bcteria.Good luck!
you can make a preculture in selective medium and the culture in non selective media.
Ummm... wait, don't the cells need to be made competent before using for transformation?
Just growing up and freezing cells in glycerol does not make them competent.
wow... I've assumed cells were previously competent and i was only a problem of making a bigger stock...
I am pretty sure if you take competent cells and grow them up to make more... all those cells that divided off the origional cells are not competent. You need to make them competent and then freeze them.
I have always understood that regular E. coli just grown in broth are not highly competent. They must be made competent by washing in cold, low salt buffers (w/ calcium chloride or rhubidium chloride), or by mixing with a solution of PEG/DMSO. See Maniatis or "current protocols" published by Wiley and Sons.
hi bluescript, fred, guys:
Yep, I made the glycerol stock of competent cells, then wondered to myself if I thawed out these frozen cells, would they retain competency? So is that the consensus- that the only way to make more competent cells is to undergo those washes mentioned?
i use regulary electrocompetent cells. to make a new stock i plate them on selective plate, do a preculture (w selection) and a bigger culture (w/o selection). then stop the culture on ice 30', pellet them, make 3 washes and a wash in 10% glycerol. Finally resuspend the cells in small volume of 10% glycerol and freeze them in liquid nitrogen and then store at -80°. Hence i assume they are ready to use because i just thaw them on ice and add DNA before electroporation.
So i assume they've been made competent by the 10%glycerol wash?
Ps : if you want the detailed protocol i use, i can mail it to you.