not able to co-transfect 293s - (Jul/11/2011 )

Dear All,

I am facing a major problem. I need to cotransfect two plasmid in 293s for CoIP study using lipofectamine 2000. however when I use my plasmids, it seems that at least one of them do not express. I have tried one of my plasmids with GFP. Sometimes GFP expresses sometimes not...

I cannot think of any special reasons. Any ideas/suggestions?

Please help

Have you tried titrating the amount of DNA and the ratio of lipofectamine:DNA you are using?

How have you prepared your DNA?

Titrating DNA would help as Bob1 said.. also why sticking up to Lipofectamine.. why not others like Fugene, if you have access to this, try out...

bob1 on Mon Jul 11 23:54:47 2011 said:

Thanks for the replies...

I use 1 ug of dna mix them well and then add 5ul lipo 2000 (for 6 well plate), mix, incubate 20min. this works perfect for 293s when I use one plasmid.

How can I titrate DNA and lipo? everytime I have to do a western blot to find out if the cotransfection has worked. do you know any easier way?

Western or immunocytochemistry/immunofluorescence would work, sorry there aren't really any easier options - you cvan at least run the titrations on the same gel. You could also look at transcript.

Are you still using 1 ug total DNA when you are cotransfecting, or are you using 2 ug total?