best oligo for EMSA - (Nov/26/2013 )
I want to synthesize an oligo 35 bp long that contains binding site for just the protein of my interest for my electromobility shift assay (EMSA). I want the rest of oligo to have no protein binding site. how can i design this oligo.
This is straightforward if you know the binding site sequence -- just surround the sequence with some junk DNA that will have little effect. Make it match the GC composition of the organism you are studying (likely around 50%). Almost all binders will only bind to dsDNA, so you will need to either synthesize the other strand, or to make it double stranded with PCR. I would probably choose to make a longer fragment, perhaps 50 bp. You could make two 35 bp oligos with a 20 bp dsDNA overlap when annealed, then do a few cycles of PCR to provide extension and a 50 bp dsDNA product.