Grenouille

Hi everyone!

I started to do a lot of FACS analysis (which is something I never did before) of viral GFP transduced cells and did my compensations by using single stained cells. I only get a small GFP signal and would like to use compensation beads to do my compensations but really don't know whether it is possible to do my GFP compensations by the use of FITC stained beads.
This might be a stupid question, but I am happy about every answer.

Thank you all!

Hi,

I just did something stupid and realized that s.th. went wrong with my cells AFTER I completely finished pipetting my calcium phosphate transfection mixes.
I don't want to throw it away !!!

Now I am wondering if it is possible to store my HeBS/CaCl2/DNA Mixes o/N @ 4°C and do the transfection tommorow.

Has anyone tried this?

So much DNA would be gone

Thanks in advance,

stupid me!

Hi,

I am planning to do an experiment in which I want to overexpress my protein of interest linked by T2A with GFP as marker. So my protein of interest and the gfp should get cleaved posttranslational.
Right now I am thinking about the best control for such an experiment. I think an empty vector (only with GFP) is better than mock but one could argue that in my construct is much more (longer) mRNA made than in the empty vector control. Because I am looking for two proteins (so two constructs) I could use one protein-construct as control for the other but we don't know enough about the proteins so I think this isn't a good idea (they could maybe work together somehow or s.th. else).

So, is there a better control than emtpy vector? Some protein-overexpression that has no effect (if something exists)? Or is my empty vector control just fine? Or is the other construct as control a good idea?

Maybe there is no perfect control...
But I just wanted to adress this question because of the highly importance of good controls.

Thanks in advance