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293T transfection - (Dec/25/2010 )

Hi All-
I have been having A LOT of trouble getting a successful transfection from a particular DNA (about 2.2kb) into 293T. My negative control and positive control transfect beautifully, so therefore I know my technique is great... but when I run a Western it appears that there is no protein being made (this DNA is flag-tagged as we are in the process of making an antibody). I was looking for suggestions to figure out if the cells are 'spitting out' the DNA and how I might go about fixing that?

Thanks in advance.


check the plasmid sequence and check if the cDNA is in frame. also is your protein secreted into media?