I'm doing miniprep in LB broth for isolating pET28a plasmid, plasmid is resistant to kanamycin, native DH5a cells are not growing on kanamycin plate.
transformed cells are growing on LB plate, and I picked some colonies and cultured in broth. But these cells from the broth after pelleting did not lyse adding lysis buffer.
(note: resuspension buffer added and vortexed thoroughly before lysis.)
can anyone solve my problem?
thank You in advance
If your lysis buffer didn't go off, if you did indeed resuspended your cells in P1 homogeneously after pelleting, then these are not Gram neg. cells, ie these cannot be your transformed E coli. Cloning strains do not alter their expression pattern too much after hosting a plasmid, let alone altering their membrane/cell wall structure.
@Ian Hu
I did transformation in same cells using ampicillin resistant plasmid and it worked well...
But I'm getting this issue only with kanamycin
@Ian Hu
But no colonies found on kanamycin plate incubating for 24 hours at 37'
- Badges
- Science topic