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GFP insert


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#1 samal90

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Posted 26 January 2010 - 02:23 PM

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

#2 scolix

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Posted 27 February 2010 - 01:21 PM

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?




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