Prepare bacteria stock - (Oct/05/2006 )
Hi, guys, normally how do you prepare the bacteria stock? in DMSO or glycerol? at what concentration? and if i want the bacteria competent for either heat-shock transformation or electrocompetent, are there any specific requirements?
To make stocks of a bacterial strain I suspend fresh bacteria, grown on plate or in culture, in a 1:1 solution of LB and glycerol.
To make chemically competent cells I use the following protocol:
Dilute 5 ml overnight culture into 500 ml LB, grow until OD600nm =0.8.
Chill 10 min on ice.
Pellet at 8000g, 4°C for 10 min.
Resuspend in 50 ml cold 0.1 M CaCl2.
Incubate in ice-water ~30 min.
Pellet at 8000g, 4°C for 5 min.
Resuspend in 5 ml cold 0.1 M CaCl2:glycerol (85:15).
Prepare 50 ml aliquots, freeze on dry ice and store at -70°C.
I'm afraid I don't have a protocol for making electrocompetent cells.
I used the SW-102 cells from NCI-Frederick for recombineering. To prepare the glycerol stock, i mixed 500ul fresh overnight with 500ul autoclaved 80% glycerol, then put the tube in isopropanal cooling box at -80C for about an hour before i finally transfered into my box at -80C. Next day, i streaked the cells on plates and found no colonies, i also tried to grow in LB media, again no cells grow... seems my bacteria are all dead. Please help me explain what happened... HELP!!!
It's a little surprising you have no growth. I use about 15% glycerol final concentration mixed directly with an overnight (no spin down). I let them sit on the bench for 1/2 hour before putting them into the -80 freezer (no fast chilling, dry ice). I've not seen this fail in many years of doing it. To recover the strain, do NOT unfreeze the sample, but pick a small part of the ice on a loop or stick and inoculate a plate. Put the frozen strain immediately back into the -80 freezer.
Thanks again, phage434! yes, it's kind of weird. i didn't unfreeze the sample, just pick a small part for inoculation and they didn't grow. For other cells, i use 3.5% final concentration of DMSO and they grow happily. I am wondering the glycerol concentration is too high, but still there are some people using this concentration, although they use flash freeze...