How do I make more competent cells? - by growing up and freezing glycerol stock? (Jun/26/2005 )

I am also a fan (and long time user) of the rubidium chloride method. I've found (as Beverly mentioned above) that I can make a ton of these up in a day, and store aliquots for long periods of time at -80°C without loosing competency. I've also found that I can just put the aliquots directly from ice into the -80°C freezer (no snap freezing with N2 or EtOH/dry ice) and they're fine.

Here's my protocol...

Hi GPCR

In the case of transformation using electroporator, you can use Fred's method. But, I am not sure if you use heat-shock method. I always use different competent cells (with different preparation) for heat shock and electrotransformation.

um, I think maybe the reason chemically competent cells can't substitute for electrocompetent cells because of the salt...excess salts will make it 'pop', and when you make chem comp cells you wash them with lots of salts to make the membrane more permeable

I typically follow the Beverly/Homebrew approach and I also use an adaptation of Inoue, but back in the day I used to electroporate fairly often, and I always had the best luck with a fresh culture...do a short O/N (~15 hrs) culture, take 1.5mL aliquots, spin and wash throughly about 5 or 6 times with ice-cold sterile H2O, (do everything on ice), after all these washes, resuspend each 1.5 mL original volume in 150 ul, use 50ul / transformation... easy to do.

with chem comp cells, I pull 'em from the -80 for around 6 months or so before I get paranoid and toss them out, and they generally work just fine

but using calcium chloride is only suitable for the preparation of chemically compitant cells... and cells you are concerned with are have to be electrocompitant.....

Hi folks,

At the risk of stating the obvious...

You have to be careful to maintain the correct selection when storing some hosts of this type, or when making more competent cells.

Some of the hosts commonly used for expression carry accessory plasmids, which are easily lost. BL21(DE3) is fine, but strains like BL21(DE3)pLysS or BL21(DE3)Codon+ have to be maintained under chloramphenicol selection. Of course these strains will grow fine without chloramphenicol, but will revert to common-or-garden BL21(DE3)...

Del.

I have a protocol for making Calcium competent cells, if you'd like it, I can email it to you. Basically, you grow up your competent cells, spin them down, and do some resuspensions in Calcium chloride.

hi

i have read in a manual for pGEX vectors , that competent BL21 cells , freshly prepared have to be used within 2-3 hours

and yes u can grow the BL21 cells in LB but they wont be competent u will have to make them competent by chemical method or electroporation
electroporation is much effective method
but i dont know if BL21 can be used for that method or not
then u can store BL21 competent cells if u immediately freeze them after making them competent and store at - 80 till further use

u will have to make into 100 micro liter aliquots in eppendorffs and freeze them immediatly in - 70 ethanol bath or liquid nitrogen and store them at - 80 or -70
and next time u use it , thaw them in ice and add the ligation mix to it and then do transformation


CaCl2 and TSS buffer both are good for making chemically competent BL21 in my recent experience

but dont make chemically competent cells to do electorporation
the salts will interfere and cause sparks in the machine
try freeze - thaw method to make competent cells in that case
thats just freezing in liq nitrogen or - 70 degree ethanol bath,, thaw in ice
then adding plasmid and freeze again ( i can send u the article if u r interested )



but i have read in the same manual that BL21 cells do not transform well
so ur transformed colonies will be lesser in numbers than when u use other cells

all the best

hi!
what about using a LB medium instead of a SOC or SOB medium when prepaing compotent cells? does it make a difference?
thanx...