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?
thx
Sam
-samal90-
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?
-scolix-