Oligonucleotides are shipped in dry (lyophilized) form and it is very easy to resuspend in an aqueous solution. Therefore, it should be dissolved only immediately before use. Modified oligonucleotides are also dissolved in sterile water at pH 7,0 or in one of the named buffers.
Take into account the following tips:
- It is very important to shortly spin down the content before opening the tube for the first time, the dry pellet could be lost if not at the bottom of the tube.
- Oligonucleotides are best stored in dry state. Generally, they are remarkably stable, even when stored at 4°C or room temperature. For longer storage, also dry oligonucleotides should be stored at -20°C.
- We recommend dissolving oligonucleotides in sterile water at a neutral pH (pH 7,0). Usually the water from water purification systems has an acidic pH, in this case, a buffer is recommended, which keeps the pH constant in a slightly basic range. We recommend using TE (10mM Tris pH 8.0; 0.1mM EDTA; pH 8.0)* or sterile water for resuspending the dry oligonucleotides.
- The use of water with a pH below 7,0 leads to depurination of the oligonucleotide. Use NaOH to increase the pH or dissolve the oligonucleotides in a suitable buffer.
- Appropriate working conditions and proper handling in a nuclease-free environment ensure a long life for RNA and DNA. This should be preferred even to DEPC-treatment. The greatest danger for dissolved oligonucleotides lies in nucleases. To minimize degradation by nucleases, store oligonucleotides at -20°C.
- Cy5-modified oligonucleotides are instable in alkaline environment. Verify if the solvent really has a pH of 7,0.
- There are benefits from aliquotting oligonucleotides into portions for immediate use and those for longer term storage.
- Adding TE or water at ten times the number of nanomoles will give a 100μM final concentration. This concentration provides 100 picomoles of oligonucleotide per μl. Most PCR reactions will use 10 to 50 picomoles of each primer per reaction.
- The most accurate means of assessing the amount of oligonucleotide present following synthesis is to measure the optical density of the final product at 260nm. This value is provided on the spec sheet and it is determined only after purification since unincorporated nucleotides and protecting groups can lead to inaccurate estimates of oligonucleotide mass.
- Working concentrations of 10 pmol/µl or less are frequently found to be unstable. Dilute only minute amounts and use only once or twice.
- Repeated freeze/thaw cycles should be avoided.
- Fluorescence-labeled oligonucleotides are principally to be stored in the dark.
