Synthetic DNA sequences, known as oligonucleotide primers, are typically delivered in a lyophilized (freeze-dried) state. This dried format ensures long-term stability and easy transport. Primers must be accurately reconstituted with a solvent to make them functional for molecular biology applications, such as Polymerase Chain Reaction (PCR). Resuspension converts the solid pellet into a liquid stock solution of a precise concentration, which is necessary for nearly all downstream laboratory work.
Selecting the Appropriate Solvent
The solvent chosen for resuspension must be sterile, nuclease-free, and chemically non-reactive with DNA. The two primary options are molecular biology grade water and Tris-EDTA (TE) buffer. Nuclease-free water is a common choice, especially for primers used quickly, but it offers minimal protection for long-term storage. Water is sometimes discouraged because its $\text{pH}$ is often slightly acidic, which can cause slow, gradual hydrolysis of the DNA over an extended period.
TE buffer is a better option for long-term stability. It consists of Tris, a $\text{pH}$ buffering agent, and EDTA, a chelating agent. Tris stabilizes the $\text{pH}$, preventing the solution from becoming acidic. EDTA binds to divalent metal ions, which inhibits the activity of nucleases (enzymes that degrade DNA). This combination offers superior protection and a longer shelf life for the primer stock. Regardless of the solvent chosen, it must be certified as nuclease-free to prevent contamination.
Calculating the Required Volume
Achieving a precise concentration in the stock solution is important for experimental reproducibility. The standard target concentration is $\text{100 } \mu \text{M}$ (micromolar). The manufacturer provides the total amount of product in the tube, usually in nanomoles ($\text{nmol}$), on a specification sheet or the tube label. Converting this mass into the required volume of solvent is a straightforward calculation based on the definition of molarity.
For a target concentration of $\text{100 } \mu \text{M}$, the calculation simplifies into a convenient shortcut: multiply the total number of nanomoles ($\text{nmol}$) by $\text{10}$ to get the resuspension volume in microliters ($\mu \text{L}$). For example, if the tube contains $\text{45.5 nmol}$ of primer, the necessary volume of solvent is $\text{45.5} \times \text{10}$, which equals $\text{455 } \mu \text{L}$. Creating a high-concentration $\text{100 } \mu \text{M}$ stock maintains the primer’s chemical stability, making it more resistant to degradation and less likely to adhere to the plastic tube walls.
Step-by-Step Resuspension Protocol
Before opening the tube, perform a quick, high-speed spin in a microcentrifuge for approximately $\text{30}$ seconds. This initial spin ensures that any primer material dislodged during shipping is collected into a single, compact pellet at the bottom. Failing to spin the tube down risks losing primer material when the cap is opened, making the final concentration inaccurate.
Once the primer pellet is confirmed to be at the bottom, accurately pipette the calculated volume of nuclease-free solvent directly into the tube, aiming for the pellet. Use sterile, filter-tipped pipette tips to prevent accidental contamination of the stock solution. After adding the solvent, the tube must be mixed to ensure complete dissolution of the pellet.
Mixing can be done by gentle vortexing or by flicking the bottom of the tube. Following initial mixing, a brief incubation at room temperature for $\text{5}$ to $\text{10}$ minutes helps the DNA molecules fully dissolve. A final quick spin is then performed to collect all the liquid from the walls of the tube, ensuring the entire volume is ready for use or storage.
Aliquot Creation and Long-Term Storage
The $\text{100 } \mu \text{M}$ stock solution is too concentrated for direct use, but it serves as the stable, long-term reservoir for the primer. To protect this stock from repeated freeze-thaw cycles and contamination, create smaller working aliquots. A typical working concentration is $\text{10 } \mu \text{M}$, achieved by performing a $\text{1:10}$ dilution of the stock solution using the same nuclease-free solvent.
For example, $\text{10 } \mu \text{L}$ of the $\text{100 } \mu \text{M}$ stock can be diluted with $\text{90 } \mu \text{L}$ of solvent to yield $\text{100 } \mu \text{L}$ of the $\text{10 } \mu \text{M}$ working solution. These small aliquots should be stored in separate, clearly labeled tubes. The concentrated $\text{100 } \mu \text{M}$ stock is best stored at $\text{-20}^{\circ}\text{C}$ or $\text{-80}^{\circ}\text{C}$ for maximum stability, extending the shelf life for two or more years. The $\text{10 } \mu \text{M}$ working aliquots are typically stored at $\text{-20}^{\circ}\text{C}$ and should be the only tubes thawed for daily experimental work.