Primer Resuspension and Dilution Calculator
Calculate primer resuspension volumes and working dilutions from vendor-provided oligonucleotide amounts. Generate stock and working primer concentrations for PCR, qPCR, RT-qPCR, sequencing, cloning, and molecular biology workflows.
Primer Preparation Setup
Enter each primer or probe exactly as supplied by the vendor. Amount is total material in the tube; stock concentration is the concentration after adding diluent.
| Primer / Probe Name | Type * | Fluorophore | Primer Amount * | Desired StockConcentration * | Action |
|---|
Optional Working Dilution
Use this when you want to prepare a lower-concentration working stock from the concentrated primer stock.
Primer Resuspension Results
Bench Setup Summary
Detailed calculation
ELN-Friendly Method Summary
Formula and Calculation Logic
Primer vendors commonly report dried oligo quantity as nmol. That value is the total primer amount in the tube, while µM describes the concentration after the primer is resuspended.
Because 1 µM equals 1 pmol/µL, a 100 µM stock equals 100 pmol/µL, or 0.1 nmol/µL. A 32.8 nmol primer resuspended to 100 µM therefore requires 328 µL.
Working dilutions use C1V1 = C2V2 to calculate how much concentrated primer stock and diluent are needed for the selected final working volume.
Example Workflow
A scientist receives GAPDH forward and reverse primers, each supplied as 32.8 nmol. The goal is to prepare 100 µM stock solutions and a 10 µM working solution for routine PCR setup.
Briefly spin down each lyophilized primer tube before opening, then add 328 µL nuclease-free water to GAPDH_F and 328 µL to GAPDH_R.
Mix thoroughly to generate 100 µM stock solutions. Label each tube with primer name, concentration, date, and diluent.
Prepare a 10 µM working solution by combining 10 µL stock with 90 µL nuclease-free water, then mix and store according to lab practice.
Common Mistakes
nmol is total primer amount in the tube. µM is the concentration after liquid is added.
One mM equals 1,000 µM, so a unit error changes the preparation dramatically.
Primer material can cling to the cap or tube wall. Brief centrifugation helps recover dried oligo before opening.
Volumes below about 1 µL are often unreliable and may require an intermediate dilution.
Dilution cannot increase concentration. Verify starting and desired concentrations.
Protect probes from prolonged light exposure and avoid unnecessary freeze-thaw cycles.
Check the vendor documentation for each tube instead of copying a previous nmol value.
Working stocks reduce freeze-thaw exposure and make routine pipetting easier.
Frequently Asked Questions
What stock concentration should I use for PCR or qPCR primers?
Many labs prepare concentrated primer stocks at 50 µM or 100 µM, then create 5 µM or 10 µM working stocks for routine assay setup.
What is the difference between a primer stock and a working stock?
A primer stock is the concentrated long-term storage solution prepared after resuspension. A working stock is a lower-concentration dilution used for routine experiment setup.
Why do vendors report primer amount in nmol?
nmol reports total oligo amount supplied in the tube. It lets you calculate the exact volume needed to create a desired concentration without needing molecular weight.
Do I need molecular weight to resuspend primers?
Usually no. For routine resuspension, use the vendor-provided nmol amount. Molecular weight is only needed when converting from mass instead of using the reported amount.
Should I use nuclease-free water or TE buffer?
Nuclease-free water is common for routine PCR and qPCR workflows. TE buffer may improve long-term stability, but EDTA can interfere with some sensitive enzymatic reactions.
Is it okay to vortex primers?
Brief vortexing is generally acceptable for standard DNA primers after adding diluent. Spin tubes down afterward. Probes and modified oligos should be handled more gently when appropriate.
Should primer stocks be kept on ice?
Short bench periods are usually acceptable for standard DNA primers, but working on ice during longer setup sessions can reduce warming and support consistent storage practice.
How should fluorescent probes be handled and stored?
Protect fluorescent probes from prolonged light exposure and repeated freeze-thaw cycles. Aliquoting and low-light storage can help preserve fluorescence performance.
How long are resuspended primers stable?
Stability depends on oligo chemistry, buffer, concentration, storage temperature, and freeze-thaw history. Many standard DNA primer stocks are stored at -20°C for long-term use.