I am trying to use DNA probe to capture long RNA sequence. I heat the RNA to break the loop and add RNase inhibitor to protect it. However, the result always show RNA/DNA binding but denature again in a short time. I am confident that the sequence is specific and conserved.
How can I prevent the melting? Do I need to add any hybridization buffer to improve it?
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A couple of things I can think of that might help. Add more GC content to your probe or make it longer. Definitely do the hybridization and subsequent storage in a buffer with some salt not just water. Perhaps 10-50mM NaCl might work, you could test different concentrations you don't want to encourage nonspecific binding.
When you heat your RNA make sure there EDTA present, it will bind up any magnesium if present, and helps protect RNA from hydrolysis during heating.
Whatever conditions you are doing for your hybridization put the RNA/DNA on an ice slurry to lock the hybridization in place. I say slurry meaning ice with water in it, it will be colder than just ice and snap chill the samples more quickly.
Good luck!
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