I have eGFP and the gene fragment i assembled by pcr.The next step is that i have to join my fragment fragment into 3'p end of the eGFP.I have three primers FP1 is forward primer for eGFP, FP2 has overlapping between eGFP and my fragment. RP1 is just revcom of my fragment 3 prime sequence. How can I assemble this by pcr. is it possible to mix all the three primers together with GFP and my fragment.
Well, technically yes but no. Why yes is because on paper this looks simple enough but think about the technicalities. Your FP1 and FP2 will have different TMs, meaning the temp required to make the complete fragment with FP1 and RP1 will be different than to the MP of FP2. Effectively when the temp for FP2 reaches it might cause FP1 and RP1 to detach.Or the opposite might happen, where FP1 and RP1 are attached whereas FP2 might not.
Another problem I could think of is that as the PCR is cycling, the difference will lead to random length amplifications if at all you get any. The maximum amp product you might see will be of Frag-2.
If you want to fuse such fragments, I suggest going for OE-PCR (Overlap extention PCR). This is a highly effective method to join two pieces of DNA using PCR. the concept is similar to what you have proposed. Look at the wiki page- https://en.wikipedia.org/wiki/Overlap_extension_polymerase_chain_reaction This image also explains the process...