I'm at my wit's end trying to overexpress a gfp-tagged protein in human breast cancer cells. I have a (purchased) plasmid with the ORF of my gene of interest. I PCR-amplified this ORF and added a kozak sequence (GCCGCC) as well as appropriate flanking restriction sites. I subcloned this into pEGFP-C1 and screened the resultant colonies. Sequencing results show that is in frame with pEGFP. I transfected the cells-- and got nothing. A transfection with empty vector worked just fine. Any suggestions for troubleshooting? one thing that has me very confused is that my protein is downstream of egfp, so how is it possible that I am not getting a signal? Thanks!
Edited by yvette9995, 02 October 2009 - 01:17 PM.