Direct observation of pEGFP from glycerol stock

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Direct observation of pEGFP from glycerol stock

Postby Mahirah » Apr 17 2005 5:47 am

Hi,

I probably going on the short-cut. I do not have much comprehension about pEGFP-N2 and reading through confuses me more... :?: I am wondering if it possible to view the flourescence glow directly under the flourescence microscope after streaking the cell (E.coli containing the plasmid) from the glycerol stock?

p/s: (Of course this is to view the single colony)

Regards,
Mahirah
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Postby kayvee » Apr 17 2005 2:00 pm

Well, you will be able to see the colony fluorescing if the GFP is expressed in E. coli. First, check which promotor is the GFP gene is under. If it is under some mammalian promotors like CMV or SV40, then you would not be able to see the expression (As far as I remember, this pEGFP vector has CMV promoter). Secondly, the E. coli cells should be of the strain where in you can express proteins (like BL21, BL-R, etc). Finally, the E. coli cells need to be induced. So, you should have IPTG in your agar media.

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Postby rxl5124 » May 03 2005 8:58 am

I think pEGFP-N2 is from Clontech and is a mammalian expression vector so you can only see green cells when you express this construct in mammalian cells. The stock you have is purly to allow you to to grow up and purify more of the DNA construct itself.
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Postby MSU_ChE » May 05 2005 5:11 pm

I am pretty sure the vector is from Clontech, as rxl5124 said. I'm actually using one of the destabilized versions of that same EGFP line. This line uses the CMV promoter, so you'll have to transfect the plasmid into a mammalian cell line before you'll see fluorescence.
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Postby 2fluffy » May 06 2005 3:46 am

Why not just try it?! It wont take long and if you do see fluorescence you will know its worked and if you dont you can just carry on with what you are doing.
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Postby skeurto » May 06 2005 12:05 pm

The vector in question is definitely a mammalian vector - I have it. The OP won't see anything.
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Postby Mahirah » Jun 01 2005 1:04 am

Thanx for all responses!

Cheers!!!
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Postby MSU-Genetics » Jun 05 2005 9:03 pm

I indeed see expression of dsred in my Ecoili stock, which is very nice, but my GFP one is not very good.
Best
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