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fusion of cDNA expressing protein with gfp

protein expression molecular cloning

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#1 ginger-black

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Posted 29 June 2012 - 02:18 AM

Hi
sorry if my question seems very basic! I really do nott have access to my protocols now and I do not clearly remember what we did before.Protein has not been my area of expertise.
I want to clone a known cDNA with Age1,Nhe1 upstream of IRES sequence so my final protein be expressed in fusion with GFP located at 3prime.here is the address of vector 26670 at addgene

That is Promoter(Synapsin)+cDNA+IRES+GFP+wpre+ltr+PA terminator

Since my full length cDNA is already cloned in another vector I just need two primer with Age1 and Nhe1 sites to amplify the fragment and clone into that aforementioned vector.the problem is that I'm not sure where these primers should start and end?I mean the first of primer should be ATG of cDNA and the end before pa signal or after that in cDNA???how I can be sure that recombinant transcript would be correctly translated to desired protein?
plz help me on this issue

#2 Curtis

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Posted 03 July 2012 - 12:11 AM

The first thing you need to do is to avoid keep saying cDNA! When a cDNA is amplified with PCR it is not called a cDNA anymore.

But I do understand your question. primer design is always a big issue. I can only answer your question briefly, and you need to find some other text books as well.

For fusion, you need to have your gene and start codon (ATG) in the same ORF with the GFP tag. Most vector manuals explain how to do this clearly. Read up your manual.

For your forward primer you definitely need to have 4-5 nt over hang (or less, look at Fermentas handbook), followed by an RE site, and then a Kozak's sequence (optional) and finally 18-24 nt your gene starting with ATG. you need to make sure the 18-24 nt binding region shares a Tm of -/+5C with your reverse primer.

For your reverse, you follow the same approach, but you need to avoid having the stop codon. If you put stop codon then your gene can't fuse with the tag.





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