1 reply to this topic

[sata]

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

[rosepolaris]

sata, on 07 December 2010 - 05:23 AM, 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.