One generally thinks of transcription factors binding to double stranded DNA. Why do you want to use a single stranded probe?? (by probe I am assuming you mean binding partner).

Yup. How to design double stranded probe containing binding site for one TF? We have to synthesized them, what conc, should we synthesized? as we have to synthesized them commercially so I have no idea about conc. 25nM or 100nM?

Do you mean 25 - 100 nanomolar concentration or 25 - 100 nanomoles? Will you be radio labeling your probe?? If you are you do not need much starting material. Note: if the probe is long I recommend acrylamide gel purification after synthesis.

I have done gel shift assays with single stranded RNA. After 5' 32P labeling and gel purification I dissolved my RNA to ~ 1 picomole/ul or 1 micromolar stock concentration, this gives ~10,000 cpm/ul. The final concentration of probe I use in my binding reaction is ~0.1 micromolar and a total of 0.5 picomoles of probe is loaded per lane. This gives PLENTY of signal after a 1 hr exposure. Assuming a 50% loss of starting material during labeling and purification 25 nMoles is enough for 25000 lanes!!

I am not labelling my probe, I am using SYBR green and SYPRO Ruby kit for EMSA.