pNL43 transfection problem - (Aug/07/2008 )
hello evrybody
i am having a problem in trasfecting pNL43WT plasmid into my 293A cells
i used the same protocol and there was no problem but suddnely itsnot working.
i am using 6 well plate/3.6x105 cells/2.5 ml media /lipofectamine2000/and after 2 days by checking supernantent p24 level by ELISA NOTHING !!!!! and ELISA is fine , i mean the standrds are ok?
i think that my cells were overconfluent after 2 days of transfection ... could this be the reseaon for the undetected level of virus?
thankx
i am having a problem in trasfecting pNL43WT plasmid into my 293A cells
i used the same protocol and there was no problem but suddnely itsnot working.
i am using 6 well plate/3.6x105 cells/2.5 ml media /lipofectamine2000/and after 2 days by checking supernantent p24 level by ELISA NOTHING !!!!! and ELISA is fine , i mean the standrds are ok?
i think that my cells were overconfluent after 2 days of transfection ... could this be the reseaon for the undetected level of virus?
thankx
Are you using the same prep of DNA as before? Have you tried a fresh batch of cells? Can you do a control transfection with a vector encoding a fluorescent protein?
hi there
yes i am using the exact protocol , with the same plasmid and i tried new cells , i will check it tomorrow by ELISA and get back to you....
hmm, I have not failed completely for 293 cells on my hands. maybe this is because some simple mistake you have made? good luck!