Unreliable Protein Induction - (Dec/12/2006 )
Hello,
I have been trying to induce overexpression of a 25kDa protein from a pBAD system in BL21(DE3) cells with mixed results. It has worked twice before but I have been unsuccessful with this latest attempt. I have been using the same media and materials all three times. The procedure I have used is also identical. The only difference between the successful and failed attempts is that I inoculated the overnight starter culture straight from glycerol stock in this latest try instead of using a colony off a fresh plate. I don't think that would account for the trouble I've been having though.
Any suggestions about what to check? The lab's determined that for this particular clone, the conditions I'm using are optimal.
Thanks.
-mrgilmorean-
QUOTE (mrgilmorean @ Dec 13 2006, 04:26 AM)
Hello,
I have been trying to induce overexpression of a 25kDa protein from a pBAD system in BL21(DE3) cells with mixed results. It has worked twice before but I have been unsuccessful with this latest attempt. I have been using the same media and materials all three times. The procedure I have used is also identical. The only difference between the successful and failed attempts is that I inoculated the overnight starter culture straight from glycerol stock in this latest try instead of using a colony off a fresh plate. I don't think that would account for the trouble I've been having though.
Any suggestions about what to check? The lab's determined that for this particular clone, the conditions I'm using are optimal.
Thanks.
I have been trying to induce overexpression of a 25kDa protein from a pBAD system in BL21(DE3) cells with mixed results. It has worked twice before but I have been unsuccessful with this latest attempt. I have been using the same media and materials all three times. The procedure I have used is also identical. The only difference between the successful and failed attempts is that I inoculated the overnight starter culture straight from glycerol stock in this latest try instead of using a colony off a fresh plate. I don't think that would account for the trouble I've been having though.
Any suggestions about what to check? The lab's determined that for this particular clone, the conditions I'm using are optimal.
Thanks.
Did you not get any expression from the last expt? What exactly was different?
If the source of cells (glycerol vs plate) is the only thing that's changed, chances are that that is the problem. There's something about cells stored in glycerol: they like to be treated nicely for a while before being turned into protein factories!
-swanny-