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Dissolving and storage

Dissolving of oligonucleotides

Dissolving of oligonucleotides recommends to dissolve 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. Use Tris-HCl, TE, PBS or TSE.

Before opening the tube for the first time, shortly spin down the content. The dry pellet could be lost if not at the bottom of the tube.

  • Modified oligonucleotides are also dissolved in sterile water at pH 7,0 or in one of the named buffers. Cyanine dyes are instable in alkaline environment. Particularly for Cyanine5-modified oligonucleotides one should take care that the solvent really has a pH of 7,0 !
  • Oligonucleotides are best stored in dry state. Therefore, they should only be dissolved only immediately before use.
  • 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 (see below).
  • The use of EDTA (e.g. in Tris) does not influence the oligonucleotide itself, but should be taken into account with regard to subsequent enzymatic reactions.
  • 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.
  • DEPC (diethyl-pyrocarbonate) treated solvents are, even in combination with following RNase-sensitive methods, not recommended due to the high toxicity. Besides a moderate genotoxicity of DEPC itself, the highly carcinogenic ethylcarbamate is formed when combined with ammonium ions, as are used e.g. in the precipitation of nucleic acids. Further DEPC modifies purines in single strands. RNA that is modified in this way will not be reverse transcribed properly (Ehrenberg, Fedorcsak and Solomosy, Prog Nucleic Acid Res Mol Biol. 1976;16:189-262, Mandiyan and Boublik, 1990, NAR 18, 7055-7062).

Storage of oligonucleotides

Storage of oligonucleotides

  • 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.
  • The greatest danger for dissolved oligonucleotides lies in nucleases. To minimise degradation by nucleases, store oligonucleotides at -20°C.
  • Repeated freeze/thaw cycles should be avoided (Davis, O'Brien and Bentzley, Anal Chem. 2000 Oct 15;72(20):5092-6). Ideally, aliquot also stock-solutions or best, lyophilise the aliquots.
  • Working concentrations of 10 pmol/µl or less are frequently found to be unstable. Dilute only minute amounts and use only once or twice.
  • Fluorescence-labelled oligonucleotides are principally to be stored in the dark.
  • Thiol-modified oligonucleotides tend to oxidise and show lower stability compared to other modified oligos.