colony growth question - (Apr/26/2004 )

Hello!

I have following problem:

On thursday evening I plated my transformants out on 50mg/ml Kanamycin-LB-plates. When checking on friday and saturday, there were no colonies visible. But on Sunday i saw suddenly really a lot (!) of small and a few bigger colonies... Thy seem to grow very slowly, since i dont see much growth from sunday to monday.
The competent cells were DH5a and the plasmid is about 14kb.

Do you think these cells are relly transformed and carring the plasmid or are there other explanations according to their strange growing behavior?

Thank you!

You may have to decide it by colony PCR on those bigger colonies because small colonies may be transformants or may be not. Some recombinant proteins are not well tolerated by E. coli and can result in poor transformation or small colonies. Make sure your Kanamycin and plates are not too old.

I concur with labrat. Smaller colonies are suggestive of compromised kanamycin either by antibiotic degradation due to age or beta lactamase action. The possibility of transformants in the current group is highly unlikely although not entirely impossible. (You mean 50ug/ml kanamycin right, and not 50mg/ml?)