Hi Pasquale,
2% efficiency is too low. you would probably need to optimise lipofectamine: dna ratio a bit more. I am not sure if you are using optimem it increases efficiency. If not then use fcs free media to transfect for a couple of hours and then topup with fcs media. I usually use about 700ul of transfection media per well of six well plate and then top it up wil two ml of whole media after a couple of hours. Otherwise you could use electroporation which has slightly better transfection efficiency. Once your transfection efficiency gets better then you can throw in the puromycin to select cotransfected cells. good luck cheers, Imran Sent from Yahoo7 Mail on Android From:"pasquale pensieri" <p.pensier...@gmail.com> Date:Sat, 7 Feb, 2015 at 6:37 Subject:co-transfection efficiency Hi all, We are working with 3t3 cells in which we want co-transfect two plasmids with Lipofectamine 3000 (Invitrogen). Now one of two plasmids is GFP positive, so we enrich positive cells with FACS. But the efficiency was of 2%. Now we are thinking if these cells will be also Puro (the other marker) positive or not. And in which percent.... Can somebody help me? Thanks _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
