fusion of cDNA expressing protein with gfp - (Jun/29/2012 )
sorry if my
I want to
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
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.