I recently cloned my gene of interested into a Pet32a vector containing GFP. I ran an expression test on colonies that were positive after running a single colony PCR but the before induction and after induction (sample taken every hr for 4 hrs). It didn’t express so I used a new foward primer with a linker and PCRed again, cloned and tried the expression test again. Still no expression..any ideas..it was suggested I could use a longer linker but maybe for now because I am running out of time I will leave it and concentrate on my other experiments…do you think 4hrs wasn’t long enough..maybe it expresses after 7hrs?? Any idea for other reasons..
What was your system for induction? At what end you have the GFP? What about trying the other end? Do you have any idea about structure of your protein?
Did you try to detect your protein or GFP with blotting?