I have performed the DEAE-dextran transfection of the EL-4 cell line for
a while, and finds it quite successful. However,when I cotransfect JNK-
and MEKK- encoding plasmids together with the reporter,I failed to see
any activation effect that ought to occur. One of the elder colleagues
mentions that the order of addition of DNA to cells affects the final
outcomes. According to her, DNA should be mixed with STBS and added to
cells before addition of DEAE-dextran (final conc: 500ug/ml in STBS).
Otherwise, the DNA would be precipitated by the DEAE-dextran, which was
no longer available to cells. However,if this were the case, I should not
observe any reporter activity that I actually finds in my CAT assay. After
scanning all the protocols available on market, I do not find any comment
on the effect of order of addition of reagents. Would anyone have comment
on this? I would greatly appreciate any advice from dear netters.
PS. Merry Christmas!
Albert, H.Y. Ho