GFP insert - trying to insert GFP in a vector (Jan/26/2010 )
hey guys, i am trying to insert GFP that I isolated from a plasmid using NotI into the same plasmid but with an E22A mutation. All my controls worked but the transformation itself didn't work. For some reason i don't have growth even with a 10:1 insert on vector ratio. I use 100ul DH5a competent cells and 1ul of my ligation. I don't really know what's wrong, my controls tell me it should work. I even tried 3:1 and 5:1 to no success.
Anyone has any ideas?
does the gfp insert have NotI on both sides? How long have you digested the DNA. Too long digestion is not good.
have you dephosphorylated the DNA?