I have thiolated oligonucleotides, which I need to reduce prior to use. Most of the protocols use DTT as a reducing agent, but there are variations in its concentration and buffer medium. Also, the stock concentration of DNA is mostly set to 100 μM when resuspending from lyophilized form. For example, a company that produces oligos suggests the use of 1X TE (Tris-EDTA) buffer and 10 mM. On the other hand, in papers, researchers mostly use phosphate buffer (PB) (usually 0.18 M, pH 8.0) and DTT concentration in a range of 50-100 mM.
After reduction, I plan to use part of the solution, and the rest to store under -20 °C. Therefore, which buffer is better for storing, and is it fine not to perform purification of the solution to remove DTT if I want just to store the solution? Of course, prior to use, purification should be performed anyway.
Can anyone give me the best practice for thiol reduction?
When reducing thiolated oligonucleotides, there are multiple factors to consider, including the choice of reducing agent, buffer composition, and storage conditions. Here's a recommended best practice for thiol reduction:
In summary, the recommended best practice for thiol reduction of oligonucleotides includes using DTT as a reducing agent, selecting an appropriate buffer (such as PB or TE), optimizing the concentration of DTT, storing the reduced solution at -20 °C, and performing a purification step if you plan to store the solution for an extended period before use.
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