How to Reconstitute and Dilute Primers to Make a Working Stock
If you’ve just received your oligonucleotide primers for PCR or cloning experiments, they likely arrived lyophilized (dried) in a small tube. Before you can use them in your experiments, you need to reconstitute them in liquid form and prepare a working stock at a concentration commonly used in the lab.
This blog will walk you through two simple steps:
Reconstituting the primers to make a stock solution
Diluting that stock to prepare a working solution
Step 1: Reconstituting the Stock Solution
Check the datasheet or label that comes with your primer. It usually tells you how much primer is in the tube, typically in nanomoles (nmol).
Most labs reconstitute primers to a 100 µM (micromolar) stock solution. To calculate how much liquid to add:
Formula:
Volume (µL) = Amount of primer (nmol) / Desired concentration (µM)
For example, if your primer tube contains 25 nmol, and you want a 100 µM stock:
25 nmol / 100 µM = 250 µL (0.25 x 1000 ul to convert from nano (n) to micro (u).
So, add 250 µL of nuclease-free water or TE buffer to the tube. Mix gently by pipetting up-and-down 10 times (do not vortex harshly), and you now have a 100 µM stock solution.
Step 2: Preparing the Working Stock
Most PCR reactions use primers at a final concentration of 0.1–1.0 µM. A convenient working stock concentration is 10 µM, so you don’t have to pipette very tiny volumes during experiments.
To prepare a 10 µM working solution from your 100 µM stock:
Simple 1:10 Dilution
Mix 10 µL of 100 µM stock with 90 µL of nuclease-free water.
You now have 100 µL of 10 µM working stock ready to use.
Label the tube clearly and store both stock and working solutions at –20°C for long-term use. Avoid repeated freeze-thaw cycles—if needed, make aliquots.
Bonus Tips
Use low-binding tubes for long-term primer storage.
Label tubes with the primer name, concentration, and date.
Use sterile, nuclease-free water to avoid degradation.
Summary
Need a refresher, you can watch the video to deepen the impression:
Primer Concentration and Quality in PCR
When setting up PCR, it’s best to begin with the primer concentration recommended in the enzyme’s protocol—typically between 0.05–1.0 µM per primer. From there, the amount can be fine-tuned depending on experimental results. Accurate primer concentration measurement is essential and can be determined spectrophotometrically using the absorbance at 260 nm together with the primer’s molar extinction coefficient (ε260).
Using too much primer can increase the likelihood of non-specific binding and unwanted amplification products, while too little primer can reduce amplification efficiency and compromise assay linearity in applications such as qPCR.
Equally important is primer quality. Oligos should ideally be desalted or HPLC-purified to remove synthesis byproducts that may interfere with PCR performance. If primer stocks are in doubt, re-measuring their concentration with a spectrophotometer provides reliable confirmation before use.
Conclusion
With these simple calculations, your primers will be ready for PCR, cloning, or any other DNA work. Keeping a 10 µM working solution in your PCR setup makes life in the lab a lot easier!
Subscribe by Email
Follow Updates Articles from This Blog via Email
No Comments