good control for transfections - (Feb/16/2008 )
hi guys, i am doing some transfections using the ptarget vector in PC12 cells and showing expression of my protein of interest via immunofluorescence microscopy. what are some good controls that are absolutely needed? right now i have an untransfected control. would an empty vector control (does not contain gene encoding protein of interest) be absolutely necessary? thank you.
Hi,
I really think you should make a transfection control with an empty vector. You never know you may find something strange, and/or a reviewer may ask you that in the future, so be prepared.
Fabien
you need a control with the same mock vector; the only difference should be the absence of the gene of interest; sometimes the resistant factors make effects...
you need a control with the same mock vector; the only difference should be the absence of the gene of interest; sometimes the resistant factors make effects...
what would be a good way of knowing if the empty vector got transfected in the cells? i can't really use gfp because my seconday antibody fluorophore also fluresces in that range? any suggestions would be much appreciated.
thanks for your suggestions so far, guys. for the empty vector control, should it just contain control DNA? if so, i won't really know if the cells actually got transfected with this because there is no marker. the only other marker that i have is GFP but that already fluoresces in the same range as Cy2, which is the fluorophore on my secondary antibody so this won't be useful to me. any suggestions would be appreciated.
you could transfect with some fluorescent protein containing plasmid for transfection control, could be gfp or even some thing red like RFP or DsRed.
RFP, CFP, YFP...
all have different colour channels.
all have different colour channels.
I would suggest doing a control for the GFP - perhaps, use FLAG. Also, you can co-electroporate your gene without GFP on the plasmid with another plasmid containing GFP, but not your gene.