Witchcraft! - (Jul/16/2001 )
I have transformed Dh5a ecoli's with pTracer-SV40 vector from invitrogen. This vector contains a Zeocin resistance-GFP fusion protein. My bacteria have becomeZeocin resistent but show no fluoresence...weird? The LB I use to miniprep turns green...maybe an GFP effect...? Now the thing comes: I am not able to isolate any DNA from my transformedE.Coli's...I put in a vector and I want it BACK! NOW! The miniprep kit i use is promega's wizard sv kit. I have tried different combinations of buffer quantity without effect. I have left out the zeocin to test whether this stuff destroyed my plasmid...but also without it there no isolation possible.. My kit works because I always take a control strain thatalways yields a vector...This is very frustrating....it should be so simple...Why is this happening to me? Anyone got a clue?
Have you checked whether the cells lost the plasmid during your overnight incubation? I hope this is not a silly question but isnt Zeocin used in eukaryotic vectors when transfected into eukaryotic cells?