emsa oligo annealing - biotin labelled oligos (Mar/04/2008 )
Hi guys,
Does anyone have a reliable protocol for 5'-biotin labelled oligo annealing?? I've been heating my oligos at 95 degrees for 10 mins and then leaving to cool at RT for 30 mins.
I've run into the problem that my gels have bands corresponding to single-stranded and double-stranded oligo.
I also have the problem of protein sticking in my wells.. i read that only using freshly made oligos may help this. Any thoughts??
Thanks,
Sara
Hi, this is my protocol for oligo-annealing, although my oligos are not biotin-labelled, but I don't think this will make a big difference:
Primerannealing
10x annealing buffer:
200 mM Tris pH 7,6
100mM MgCl
500 mM NaCl
Annealing reaction:
50 µg Oligo 1
50 µg Oligo 2
26 µl 10x Annealingpuffer
134 µl TE
Heat for 5 Minuten at 70°C, let the oligos cool down to room temperature over night in the heater. They have to cool very slowly, otherwise they may seperate again. I always put a heap of paper cloths on the heater to isolate them a bit more for a real slow cooling. Next morning you can run a gel and see if they are all annealed.
You'll always get some single stranded oligos, unless you get the stoichiometry exactly right. You could try adding BSA to your protein solutions to reduce binding of your protein to the well. I always anneal by putting a sealed screw cap tube into a large beaker of boiling water and then letting the beaker cool to room temperature. If you like using expensive equipment, you can do the annealing in a thermal cycler also.
Primerannealing
10x annealing buffer:
200 mM Tris pH 7,6
100mM MgCl
500 mM NaCl
Annealing reaction:
50 µg Oligo 1
50 µg Oligo 2
26 µl 10x Annealingpuffer
134 µl TE
Heat for 5 Minuten at 70°C, let the oligos cool down to room temperature over night in the heater. They have to cool very slowly, otherwise they may seperate again. I always put a heap of paper cloths on the heater to isolate them a bit more for a real slow cooling. Next morning you can run a gel and see if they are all annealed.
Thanks for that. To be honest, I'm a little reluctant to add salt to me mix, I'm not sure of how it might affect binding to my protein of interest (if it will affect it at all). I've tried re-annealing by heating at 95 degress for ten mins in a heating block and then allowing the oligos to cool overnight, while still in the block. It appears that I still have some single stranded oligo.
Thanks for your advice. I seem to have gotten rid of the protein that was stuck in my well. I did this by decresing the volume of loading dye that I use and by spending a bit of time making sure that each well has no residual acrylamide stuck in it. I'm also filling eash well with my buffer and then emptying it again with a gel-loading tip. This seems to do the trick.
As for my oligo annealing, I'm attempting to combine my non-labelled to labelled oligo in a 1.1:1.0 M ratio. Maybe I should increase the concentration of my non-labelled oligo as I still have single stranded oligo even after allowing the oligos to cool overnight.
If you don't have equimolar amounts of the two oligos, you will always have single stranded forms of the oligo in excess. If you mix equimolar amounts and anneal, then you should get little single stranded results.