I received an E. coli miniculture (not frozen, cooled with a cooling bag) sent by an international collaborator. (One day long travelling)
The day I received it, I inoculated into LB-Amp medium and then next day I made a STET and a Wizard miniprep. Both contained the correct plasmid. (Checked by test digest and agarose gel electrophoresis.)
The same day I made a glycerol stock. Just measured glycerol and E. coli together, vortex and quickly to -80°C. (15% glycerol)
Two days later I inoculated this glycerol stock. It grew in LB-ampicillin, but after STET miniprep it contained no plasmid. How is it possible? The ampicillin is stored at -20°C, it shouldn't degrade.
The same day I retransformed the first STET prep into another E. coli strain and next day it grew on the plate, but the colonies were very small.
My questions are:
- is it possible that an E. coli culture grows in Amp without selection plasmid?
- what can be the problem in the process (in the original strain, in the glyc culture, in the STET prep? )