why does empty EGFP vector has no fluorescence? :/ - (Nov/27/2006 )

i am already quitting on this, but it is torturing me to not know why. I had EGFP vector from my freind. It had her insert so I cut it out. I blunt end religate, get several colonies and check colonies by RE digest. The surprising thing is tha each colony is slightly different from each other by size. I ask here on the forum and I am told it's ok just use the one that seems most correct. So I do that and clone my insert into it. So far so good. I transfect into HEK no fluorescence. I check my empty religated EGFP, no fluorescence. I check my freind's EGFP, fluorescence is fine.

Please can someone suggest some idea of why on earth empty EGFP vector won't shine?!?!? it's not the problem of frame of course.

Hey..did you check for the presence of KOZAK sequence in ur vector?!

Is there a promoter remaining in the vector?

but i am only playing with multiple cloning site. that should not effect those.

I dont know what vector you're using, but you need to be really clear on what your friend did to the vector. Any number of things could have happened when you cut the insert out, perhaps filling in overhangs has disrupted the promoter. Simply sequence the region in question to find out whats going on. What about your insert? Is it in frame with the EGFP? Sequence and find out.

Hi,

Some mammalian expression vector don't include Kozak consensus sequence. Most of the time, you'll have to include them, unless the EGFP is at N-term.

If the EGFP is at the C-term, chances are, there's no Kozak/ RBS sequence (CGCCACC ATG G) there.

Hope this help