I have done some PCR fusion reactions to create transgenics. In addition to the band that I wish to see, I always see a band close to 4kb. Is this the gfp dimer as described in the paper from Hobert lab or some shorter product that is not the full extended verison? Any advice/insight would be helpful.
Also, how would one make transgenics to express gene of interest under heterologous promoter using this approach?? I believe it would have to be a three PCR fusion reaction with one reaction of promoter, second of the gene of interest and third of the gfp?
I sometimes get non-specific bands but not frequently. I tend to just ignore them and treat it more like filler DNA in my injection mix. I amplify my targets using Roche’s Long Template Expand and TaKaRa ExTaq for the fusions.
I’ve never mixed three things together at once like that for a fusion reaction.
What I normally do is take my previously generated reporter PCR fusion and use it as a template to amplify a gene::GFP PCR product. I then take this gene::GFP PCR product and use that in a PCR fusion reaction with a different promoter (for the promoter fragment you will have to design the reverse primer complementary to the beginning of your gene of interest).