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..
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fusion protein not expressing
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