blue/white selection - (Feb/19/2008 )
hi, i transformed some e. coli with maxi prep'd DNA and got all white colonies after plating. i picked a single colony for another maxi prep. however, now that i look at my plates, all the colonies have a very faint blue color to them (not dark blue). i got about 5 ug/ul DNA after the maxi prep but this was before i noticed the faint blue. it's only been about 2 weeks since i plated them. any idea what happened? i haven't been able to test out the new DNA yet.
I might be missing something here. When you say "maxi prep'd DNA", do you mean maxi prep'd plasmid? If you had your plasmid already in E. coli, why did you have to transform it again? And why do you need blue/white selection for a plasmid that doesn't come from a ligation, but from a plasmid prep? Are you sure that the E. coli strain you're using is compatible with blue/white screen? If not, it's very likely that all your colonies will turn blue. And lastly, check the pH of the growth medium, because this could also result in blue colonies.
yes, i was referring to the maxi prep'd plasmid (which contains gene of interest). my stock plates were too old so i had to retransform some more e. coli using that maxi prep'd vector DNA.
i did some rapid cycle pcr on those slightly blue colonies, and it turns out that the expected signal is there still. so it's not just empty vector that was transformed into the bugs.
Sometimes colonies turn blue after storage in the fridge. If you transformed with maxi-prep plasmid, you can be fairly confident that it contains your insert.
Also, some blue/white selection plasmids can produce white, blue and light blue colonies depending on orientation and nature of the insert.
All in all, if you screened by PCR and got your expected amplicon, I wouldn't worry about it too much!
they turn faint blue after a while.
check google for the explanation of this. heaps of info available.
V