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Two cloning and expression problems - (Jul/31/2005 )

hi, recently I got 2 big problem with my experiment which delayed my committee meeting a lot. So I really need some help from you guys.

Fisrt, I have a gene (~1.3kb) that is already in pCAGGS vector with myc-tag (introduced by PCR primer). There is an interesting 300bp conserve domain at the N terminal (from ~20-300bp). I tried several times to clone this particular domain but never made it. Different primer, different PCR cycles...I can clone another fragment that contain this domain. But I need the exact domain which is important for the gene's function. What should I do?

Second, I successfully cloned a gene in pCAGGS vector with N- terminal myc-tag which was introduced by PCR primer. Sequence is good, no stop codon, no bad mutation...but I can not detect the expression by western blot when I transfected it into COS7 cells. But When I transfected another construct that I put GFP at the C-termianl, I can see very few cell express very little GFP. And When I stained the cells which got the construct with myc antibody. I can aslo see very low expression. The gene is mamalian gene and should not be toxic one. What is the problem?

I really greatly appreciate your help!!


any suggestion?


try first to transfect your plasmid in other type of cells (like the rat labs 293 or HeLa...).
how did you prepare your 300bp dna for cloning?