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Dissolving and storage
 

Dissolving of oligonucleotides

Dissolving of oligonucleotides

 

biomers.net 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.
 

Annealing of oligonucleotides

Annealing of oligonucleotides


Oligonucleotides are always produced as single-stranded molecules. However, for many applications, oligos are required as double-stranded molecules. 

The hybridisation of two short complementary oligos is easy and actually works „by itself“:

  • dissolve oligos to equal molar concentrations
  • mix the same volumes
  • heat to 95°C for 2 min
  • slowly cool down to room temperature
     

In order to get an optimal result, it is helpful to consider some further details: 

1.Material

The presence of salts is essential for the formation and maintenance of double-stranded DNA or RNA in aqueous solution.
Since oligos usually contain already enough salt (mainly in form of Na+), short, unmodified oligos can be dissolved directly in pure water and will hybridise successfully.
The addition of further or different salts as well as a buffered solution can be helpful. The recipes for annealing buffer vary from 10 mM Tris, pH 7,5 to 100 mM potassium acetate, 30 mM Hepes-KOH (pH 7,4), 2 mM magnesium acetate or 75 mM KCl, 20 mM Tris .
However, since many salts, especially divalent ions, may influence subsequent methods, e.g. DNA polymerases, the addition of complex salt mixtures for annealing needs to be carefully  considered.

2.Method

For a successful and complete hybridisation, it is necessary to use equal amounts of both strands. For this purpose, the two oligos are adjusted to the same molar concentration (e.g., 100 pmol/μl), and then mixed with equal volumes.

Example:
Concentration of the single strands: 100 pmol/μl, 50 μl each for annealing - You get 100 μl solution with a concentration of 50 pmol/μl double strand.

      Oligo           Amount  [µl]              Concentration [pmol/µl]
Oligo 1 50 µl 100 pmol/µl 
Oligo 2 50 µl 100 pmol/µl
Duplex 100 µl 50 pmol/µl


Basically, hybridisation of two strands just happens 'by itself'. In order to melt internal secondary structures and to give the molecules sufficient "kinetic energy", the annealing batch is first heated to 95°C for about 3-5 minutes. For short oligos, 2 minutes are certainly sufficient.

In order to give the oligos sufficient time to find their complementary sequence and hybridise completely, it is optimal to allow the batch to cool down slowly to room temperature for several hours. Ideal is a simple water bath (or a heating block), which is switched off and cools down slowly in closed state.
Alternatively, a thermal cycler can also be used. Here, too, the cycler can be switched off after heating.
Until further use, the oligo duplex is best stored at 4°C.

 

Too much effort for you?

On request, we will be glad to provide annealed oligonucleotides for you.
Please use our
Excel order form and indicate in the annotation field which of the oligos should be hybridised with each other.

Unless otherwise stated, the annealed product is always supplied in dried state.
The duplex is ready to use after dissolving in sterile water.

We are always at your disposal for further details and any questions concerning oligos:
Tel +49 731 70 396 0   I   info@biomers.net