EMSA - Probe Annealing problems (Dec/07/2010 )
Hello,
I am doing EMSA first time.My probes are 50nt.Can you please tell me how to anneal the probes? I am using 50 mM tris,10 mM NaCl & 1mM EDTA and then heat for 5 min at 95C & cool slowly on heating block.But it doesn't work I cant see annealed probes.There is no difference between annealed and unannealed probes when i run it on 0.7 % Agarose gel.Can anyone plz tell me the procedure for annealing?
Thanks
-sata-
sata on Tue Dec 7 13:23:18 2010 said:
Hello,
I am doing EMSA first time.My probes are 50nt.Can you please tell me how to anneal the probes? I am using 50 mM tris,10 mM NaCl & 1mM EDTA and then heat for 5 min at 95C & cool slowly on heating block.But it doesn't work I cant see annealed probes.There is no difference between annealed and unannealed probes when i run it on 0.7 % Agarose gel.Can anyone plz tell me the procedure for annealing?
Thanks
you may want to try a 2% agarose gel or native protein gel to see the difference
also, is your DNA palindromic? It might fold back on itself.
-rosepolaris-