Re: [DIYbio] Re: Excitation Spectra for Wildtype GFP?

Actually I find the excitation /emission properties of wt GFP easier to work with in home/garage settings. I use UV LEDs for excitation, they have emission in 390-405nm range, ie exactly where you need them.  You can even buy ready to use UV LED flashlights for less than 10 bucks. For imaging I use green or yellow photographic filter, sturdy tripod and long exposure. I also find using old manual-everything lenses well fit for the purpose, because they don't reset when you need to reset your camera for changing batteries/card etc. 

here is one example

http://www.flickr.com/photos/morinkovo_fotky/3757039367/in/photostream

The excitation and emmision peaks are sufficiently far from each other (395-509 nm) , that you can easily block all the excitation light from your  image with inexpensive set of filters. It is much more difficult with most of the engineered version with 488ex/509em peaks to filter off the blue light from your image. You might need a dichroic mirror or some really good filter, which can be expensive. 

As fir the patent issue - are you really sure that eGFP is protected for your purpose while the wild type is exempt? I am not familiar with this particular issue, but most of the time all sequences hybridising with patented sequence or with more than say 90% protein sequence identity are covered by the patent. 

good luck tomas

Dne čtvrtek, 22. listopadu 2012 15:26:39 UTC+1 Cathal napsal(a):

Hey all,
I'm going to be working with a construct containing wildtype GFP, with
the original peptide sequence from A.victoria, and I'm hoping someone
here would know whether it's feasible to excite with Blue light (which I
think you can do with normal GFP?) or if I'll have to use UV?

Thanks
Cathal

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