Storage of oligonucleotides


It is very important to store oligonucleotides under the correct conditions to avoid degradation and loss of valuable samples.

Oligonucleotides in solution

Oligonucleotides are not completely stable in aqueous solution as acid-catalysed depurination will slowly occur (The pH of distilled water can be as low as 5.5). For this reason oligonucleotide solutions should be frozen for storage. However, repeated freezing and thawing gives rise to shear stress in oligomeric molecules, and causes degradation. The best compromise is to aliquot the oligonucleotide into several tubes and to freeze the ones that are not required for immediate use.

Protocol for aliquotting oligos

On receipt of the oligo (in solution in deionized water), the solution should be divided into several tubes (aliquots), and these stored in a freezer at -20 °C. The volume of the aliquots may be 250 μL, 100 μL or smaller, depending on how many tubes you are able to aliquot. For UV melting studies large aliquots (250 μL) are suitable, while for PCR applications small aliquots (100 μL or smaller) are better. Use the same kind of tubes for these aliquots as those in which the oligo is provided (some tubes are non-sterile and can cause oligos to degrade). Each tube should be carefully labelled.

When the oligonucleotide is required, take one aliquot from the freezer (and label the tube with the date on which it was removed from the freezer). Store this tube in a fridge at 4 °C. Use this tube for about 2 weeks (and certainly no more than 4 weeks). Do not freeze it again but always keep it at 4 °C when it is not in use. After using it up (or after its “use by date”) take another tube from the freezer and start the process again. This protocol avoids repeated freezing and thawing which slowly degrades oligonucleotides.

This procedure should prevent the degradation of oligonucleotides.

Stability of DNA and RNA in solution

The chemical stability of oligonucleotides is good in buffers in the pH range 7.0 to 8.5, but contamination with micro-organisms is surprisingly difficult to avoid in an open laboratory. If this happens rapid degradation occurs. This emphasises the necessity to work in a sterile environment when manipulating DNA. RNA is much more sensitive, as RNAse enzymes are abundant on the skin; and great care must be taken when purifying RNA.

Oligonucleotides as lyophilised (freeze-dried) solids

Freeze-drying (lyophilising) involves freezing an aqueous solution of the oligonucleotide in liquid nitrogen and placing it under high vacuum on a freeze-drying machine. This removes water by sublimation and leaves the oligonucleotide as a fluffy white powder.

Owing to the hydrophilic ("water-loving") nature of DNA, many water molecules are retained after freeze-drying, by hydrogen bonding to the bases and sugars. This is a fundamental property of nucleic acids and a freeze-dried solid oligonucleotide will contain approximately 40% by weight of water (e.g. 5 mg of solid oligonucleotide will only contain approximately 3.5 mg of DNA). It is therefore not possible to accurately determine the molar quantity of a freeze-dried sample of DNA by weighing it; in order to determine the amount of DNA present, the oligo should be dissolved in water and the absorbance measured.

Oligonucleotides can be stored for long periods as freeze-dried solids.

See also

Our free online Nucleic Acids Book contains information on all aspects of nucleic acids chemistry and biology.