emsa competition oligos - how to make up competition oligos (Mar/03/2008 )

hi,

I'm new to EMSA and am having a few problems. I have run my gel and seem to have nice shifted bands.. however, the problem lies in my competition oligos. I'm not sure if I've made them up correctly.

I made up 90um stocks of probe and competition oligos, and then anneal. I find that the 1/25 or a 1/50 dilution of my double-stranded labelled probe works best.

So, if I want to add in a 25X molar x/s of double-stranded competitior, I just add 2ul of my undiltued cold probe to 1ul of my 1/25 labelled probe. Is this right?? So, if I want to add my cold probe in 50x molar excess, I just use 1ul of my 1/50 probe dilution and add 2ul of my double-stranded cold probe??

I'm afraid that any disappearance of band that I see is due due my diluted probe.. and not due to competition.

ANY help will be greatly appreciated.

Thanks,

Sara

Cold competition is relative to the amount of hot probe you use.
For example, if you use approx. 50 femtomol of hot probe you would
add 25 picomol of cold probe as a 50x cold competition.

thanks for that mikew.

I suppose my main concern is whether or not the hot probe concentration must remain constant for the various cold probe excess concentrations.

In other words, if you're adding cold probe in 25X molar excess and 50X molar excess; does the hot probe concentration for these two reactions have to be constant, or is the only important parameter the ratio between hot and cold probes for individual reactions?

I really appreciate your help. Many thanks,

Sara

Yes, use the same amount of hot probe in all reactions. The hot probe concentration will be the same.
No matter how much cold probe you add, the amount of hot probe (lets say 50 femtomoles /20 microliter rxn)
will be constant. The final volume stays constant.