Primers are short, synthetically produced DNA sequences that serve as starting points for DNA synthesis. They play a fundamental role in various molecular biology techniques by providing a specific binding site for DNA polymerase enzymes to initiate replication. Primers are generally shipped in a lyophilized, or freeze-dried, state to ensure stability during transport and storage. Proper resuspension of these dried primers is crucial for accurate and reliable laboratory results.
Essential Materials
Successfully resuspending primers requires specific materials to maintain their integrity and prevent degradation or contamination. Lyophilized primers arrive in small, sealed tubes, often with a visible pellet or film at the bottom. Molecular-grade water is the preferred solvent for resuspension because it is free of nucleases, enzymes that can degrade DNA. Alternatively, TE buffer (a solution containing Tris-HCl and EDTA) can also be used; Tris-HCl helps maintain a stable pH, while EDTA chelates metal ions that could promote nuclease activity.
Accurate liquid handling is achieved using micropipettes for precise measurement and transfer of small volumes, coupled with sterile, disposable tips to prevent cross-contamination. Microcentrifuge tubes are necessary for containing the resuspended primers. A vortex mixer is used for thorough, yet gentle, mixing to ensure the primer dissolves completely. A microcentrifuge is important for briefly spinning down the primer solution, collecting all the material at the bottom of the tube.
Step-by-Step Resuspension
Upon receiving lyophilized primers, inspect the tubes to ensure the cap is securely sealed and the dried primer material is visible, usually as a small pellet or film at the bottom. Before opening, briefly centrifuge the primer tube for 5-10 seconds at low speed (e.g., 2,000-5,000 x g) to ensure all dried material settles at the bottom. This prevents loss of primer adhering to the cap or sides of the tube.
To achieve a common stock concentration, such as 100 micromolar (µM), calculate the required volume of resuspension buffer based on the nanomoles (nmol) of primer provided by the manufacturer. For example, if a primer is 25 nmol and you aim for a 100 µM stock solution (0.1 nmol/µL), add 250 µL of resuspension buffer (25 nmol / 0.1 nmol/µL). Carefully add the calculated volume of molecular-grade water or TE buffer directly into the tube.
After adding the buffer, gently vortex the tube for 5-10 seconds to thoroughly mix the solution and aid in dissolving the primer. Following vortexing, briefly centrifuge the tube again for 5-10 seconds to collect all liquid at the bottom and eliminate any aerosols or droplets adhering to the tube walls. Allow the resuspended primers to sit at room temperature for 10-15 minutes to ensure complete dissolution, as some primer sequences may dissolve more slowly.
Determining Primer Concentration
Knowing the precise concentration of your resuspended primer is important for consistent and reproducible experimental outcomes. Primer manufacturers typically provide the synthesis yield in nanomoles (nmol). This nmol quantity is the most direct way to determine your stock concentration after resuspension. By dividing the total nanomoles of primer by the volume of resuspension buffer added (expressed in microliters), you can confirm the resulting micromolar concentration.
For instance, if you resuspended 25 nmol of primer in 250 µL of buffer, the concentration is 0.1 nmol/µL, which translates to 100 µM. While less common for routine lab work, spectrophotometric methods, such as using a Nanodrop instrument, can provide an independent verification of primer concentration. These devices measure the absorbance of light at 260 nm (A260), which is directly proportional to nucleic acid concentration. However, calculations based on the manufacturer’s provided nmol quantity remain the primary and most practical method for confirming concentration in most laboratory settings.
Storing and Using Primers
Proper storage of resuspended primers is essential to maintain their stability and prevent degradation. For long-term storage, aliquot the stock primer solution into smaller, individual tubes and store at -20°C. This practice minimizes freeze-thaw cycles on the main stock, which can degrade primer integrity and reduce effectiveness. Repeated thawing and refreezing can lead to the formation of primer dimers or other secondary structures that interfere with subsequent reactions.
For short-term use, primers can be stored at 4°C for several weeks without significant degradation. When preparing working dilutions, accurately dilute the concentrated stock solution using molecular-grade water or TE buffer to the desired concentration, typically ranging from 10 µM to 50 µM. Always use sterile techniques and materials to prevent contamination that could compromise the primer’s function.