EMSA Using Biotin labeled DNA Troubleshooting

[xliburnham]

Hello,

does anyone use the biotin labeled DNA to do EMSA?

I have one question for you. How do you get the labeled DNA?

I synthesized them by oligo company. They are 49bp in length and labeled at 3-end, and purified by HPLC. then, I annealed them according Pierce protocol. Before annealing, I measure the concentration of every single strand by NANA DROP.

after diluted, why is there big difference between different fragments at same amount?

Is there someone who can help figure out what is wrong with the procedure?

thank you in advance!

[admin]

Hello, yes DNA labeled with biotin works well with EMSA.

I usually synthesize them myself using biotin-labeling kits however oligo synthesizing companies should make them sufficient for EMSA.

I am not sure what you mean by a big difference between fragments of the same amounts. Do you mean binding of protein or gel bands of probe or concentration?

I would first check your concentrations with UV spec or run them on a gel to see if they are all the same. Did the oligo company give you concentration/amount of oligo synthesized?

This (the oligo companies mass/concentration value) may be a wrong value, you may have diluted them with less/more water/buffer, or you may simply had pipetting errors.