Understanding Resuspension in Chemistry
Resuspension is the process of redistributing particles or molecules that have settled or aggregated back into uniform suspension throughout a solution. In molecular biology, this is essential when working with precipitated oligonucleotides, proteins, and cells that have been lyophilized or frozen.
When oligonucleotide synthesis is complete, the oligos are typically delivered as a dry pellet. Before they can be used in reactions, they must be dissolved into a homogeneous aqueous solution. The quality of this initial resuspension directly impacts downstream applications:
- PCR reactions — primer concentration affects amplification efficiency and specificity
- DNA sequencing — uneven oligo distribution causes variable signal intensity
- Gene synthesis and assembly — accurate concentration ensures stoichiometric ratios
Oligonucleotides are hygroscopic and sensitive to nuclease contamination, hydrolysis, and oxidation. Resuspending them in an appropriate buffer—typically TE (Tris-EDTA) or nuclease-free water—stabilizes the molecules and maintains usable stock solutions for months or years when stored correctly.
The Resuspension Volume Equation
To prepare your oligo stock solution, you need to know how much diluent (buffer or water) is required. The calculation accounts for the supplied amount of oligonucleotides and your target working concentration.
In the formula below, the factor of 1000 converts nanomoles to micromoles, matching the standard units used in molecular biology:
Volume of diluent (μL) = [Amount of oligo (nmol) × 1000] ÷ Desired concentration (μM)
Amount of oligo— The quantity of oligonucleotides supplied, typically expressed in nanomoles (nmol). This is provided by your oligo vendor on the synthesis report or vial label.Desired concentration— Your target working concentration in micromoles per litre (μM). Common choices range from 10 μM for long-term storage to 100 μM for routine use.Volume of diluent— The total volume of buffer or nuclease-free water required to achieve the target concentration. This is measured in microlitres (μL).
Buffers and Media for Oligo Resuspension
The choice of diluent significantly affects oligo stability and function. The most common resuspension media are:
- TE buffer (Tris-EDTA) — pH 8.0, contains Tris for buffering and EDTA to chelate metal ions that can catalyse nucleic acid degradation. Standard choice for long-term storage.
- Nuclease-free water — suitable for immediate use or short-term storage, but lacks buffering capacity. More susceptible to pH drift.
- Phosphate-buffered saline (PBS) — maintains physiological pH and osmolarity; preferred when resuspending cells or proteins alongside oligos.
- Custom buffers — some applications (e.g., rolling circle amplification or in vitro transcription) benefit from salt concentration, MgCl₂, or BSA tailored to the downstream reaction.
Always use sterile, nuclease-free diluents. Even trace nuclease contamination will degrade your oligo stock over time, rendering it unsuitable for sensitive applications.
Cell and Protein Resuspension Considerations
Beyond oligos, resuspension applies to cells harvested from culture and protein precipitates. Cell resuspension buffers differ structurally but serve the same purpose: uniform redistribution in aqueous medium.
A typical cell resuspension solution contains:
- A buffering agent (phosphate, Hepes, or Tris) to maintain pH
- Salts (NaCl, KCl) to match osmolarity and prevent cell lysis
- Cryoprotectants (DMSO, glycerol) if cells were frozen
- Optional nutrients (glucose, amino acids) to support short-term viability
Resuspending adherent cells or clumped pellets requires gentle agitation—vortexing too vigorously can cause mechanical damage and reduce cell viability. Pipetting up and down or brief sonication is often more effective.
Common Pitfalls in Oligo Resuspension
Accurate resuspension requires attention to detail and proper technique.
- Incomplete dissolution of pellet — If the pellet does not fully dissolve, the actual concentration will be lower than calculated. Allow resuspending oligos to stand at room temperature for 30 minutes, then gently vortex or pipette repeatedly. For stubborn pellets, warm to 37 °C briefly, but avoid heat above 50 °C, which degrades nucleic acids.
- Contamination with nucleases — Nucleases are ubiquitous in skin, dust, and even commercial buffers if not labelled nuclease-free. Use only certified nuclease-free pipette tips, tubes, and diluents. Change pipette tips between samples, and never touch the internal surfaces of tubes or pipette tips with bare hands.
- Incorrect unit conversion or calculation — The most frequent error is forgetting the 1000-fold conversion factor between nanomoles and micromoles. Double-check your supplier's nmol value and your desired μM concentration before calculating. Using this calculator eliminates manual arithmetic mistakes entirely.
- Evaporation and storage degradation — Oligo solutions stored in non-sealed or partially sealed containers will concentrate as solvent evaporates, skewing your concentration assumptions. Always use sealed, sterile tubes and store at −20 °C or −80 °C for long-term use. Even in TE buffer, oligos can be damaged by freeze-thaw cycles; divide large aliquots into smaller working stocks to minimize repeated thawing.