10 replies to this topic

[Tracy0110]

I have constricted a pGreen plasmid to contain my DNA of interest and cloned the vector into e.coli. I was wondering what methods I can use to screen for GFP action in the transformed colonies on the plate other than with PCR screening?

[Curtis]

if your vector is expression vector then you must be able to see green colonies in dark.

[bob1]

PCR won't tell you if your plasmid has activity, just whether there is plasmid there or not.  Restriction digest will/should do the same.  Sequencing will also tell you if the gene is there, but activity can only be assessed by looking for the GFP expression either by it fluorescing or on a western blot using anti-GFP antibody.

[Trof]

Actually I had (mammalian though) vector with bicistronic GFP and I just wondered If I'll see the bacteria green, but they were all green, they're a  bit fluorescent on their own.
Of course the expression from CMV promoter is much lower in prokaryots, so it may be different with bacterial promoter and probably brighter then.

[pDNA]

activity of CMV promoter in bacteria is ZERO!

Regards,
p

[Curtis]

one thing I never understood was why bacteria expressing GFP glow in dark, but to view eukaryotic cells expressing GFP you need to use fluorescence microscope? why don't they glow in dark too?

[leelee]

Somebody else said that to me, and I didn't believe them , do they really?
I've always thought that GFP requires UV/blue light to exhibit fluorescence?

[Curtis]

we even extracted the GFP from bacteria, they really glow in dark.

[phage434]

GFP is a protein.  It is fluorescent.  It does not glow by itself.  Ever.

[Curtis]

Then how do some insects glow green at night?

[phage434]

Bioluminescence is completely different from fluorescence.  Things that glow all by themselves are bioluminescent.  There are several chemically distinct types of bioluminescence.  I'd recommend you look at the literature on insect bioluminescence.

Some bioluminescent systems use fluorescent molecules to alter the color of the bioluminescence.  GFP was isolated from a bioluminescent organism, where it converted blue light to green light.