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[abc68]

hi, dear all

~9.6kb recombinant plasmid was transformed into DH5alpha cells plated on the LB/Kan (60ug/ml), the positive control turns out  in 16 hrs, while the negative control in 20 hrs,  however the recombinant clones appear after 24 hrs, and grow very slow, but they are equally spread. now 48 hrs past, the clones are no more than 1 mm. my question are: if these clones are possibly Kan sensitive or if the insert generat toxic material which impeding cell growth.

best,

[franck]

Welcome to the strange land
I have the same pb, but i use a different medium, then i can say that is no the medium. This pb is less important when selection is done in liquid medium. If you add quickly the anitbiotic and cells, they are directly dead, you must wait 2 days before added cells, during this time the product can penetrate in the gelose. Perhaps is it that?