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Suddenly no colonies, can cells 'go off' in -80? - (Jun/12/2008 )


I've got a few years of cloning under my belt now, but am stumped with a problem at the moment. We use DH5alpha cells for standard transformations of ligations and whole plasmids, for minis midis etc, and its been working fine for as long as i can remember. But recently our ability to transform has been really patchy, i.e. I've tried transforming whole plasmids uncut and I get a few colonies, but when transforming ligations theres nothing, even on control plates of just cut vector with and without ligase. We've tried changing the plates, different antibiotic resistances, ligation buffer and enzyme, have used plasmids I've used to clone with before without problems, everything. The only thing i can think of is its the cells we use (which we make competent ourselves), but they were tested when we made them about a month ago and they were fine. Other members of the lab sometimes get colonies, but its hit and miss. Is it possible for cells to go off in the -80? Or to have bad aliquots of cells in a single batch? And why would whole plasmid transformation be ok, and ligations not? I used to get an ok background of colonies on my control plates but now nothing grows. Any ideas?




Have you checked your SOC recovery medium? It can easily become contaminated and worthless. Check the temperature of your water bath and incubator (with a real thermometer). What's your water source? Do you have new people working in the lab that might have left the cells out of the freezer? You should probably just remake the cells and test them again to isolate this problem.