How to Dilute PCR Primers: 100 µM Stock to 10 µM Working Solution

Q: How do you make a 10 µM primer working solution from a 100 µM stock?

Perform a 1:10 dilution: add 10 µL of the 100 µM stock to 90 µL of nuclease-free water or TE buffer. The result is a 10 µM working solution.

Q: How much primer do I add to a PCR reaction?

Most PCRs use 0.1–0.5 µM final primer concentration. With a 10 µM working stock, adding 1 µL to a 20 µL reaction gives 0.5 µM, and 1 µL to a 50 µL reaction gives 0.2 µM.

Quick answer

Primers arrive lyophilised (freeze-dried). First resuspend them to a 100 µM stock: multiply the nmol on the tube by 10 — that is the microlitres of buffer to add (e.g. 25 nmol → 250 µL). Then make a 10 µM working solution with a 1:10 dilution: 10 µL stock + 90 µL nuclease-free water. Use the working stock for PCR; keep the 100 µM stock frozen as your master copy.

Why two concentrations?

Here is the thing most protocols gloss over: you never pipette straight from the master stock into a PCR. Every freeze–thaw cycle and every pipetting degrades the stock a little and risks contamination. So the standard practice — used in virtually every molecular biology lab — is a two-tier system. The 100 µM stock is your precious master copy: it lives in the freezer and you touch it as rarely as possible. The 10 µM working solution is what you actually pipette from day to day. When it runs out, you make a fresh one from the stock. Simple, and it keeps your primers good for years.

Step 1: Spin down the tube

Before you open anything, give the primer tube a quick spin in the microcentrifuge — about 10 seconds. The lyophilised pellet often gets dislodged during shipping and ends up in the cap. Skip this step and you can lose half your primer the moment you pop the lid. We have seen it happen more times than anyone admits.

Step 2: Make the 100 µM stock

Check the tube label or the specification sheet for the primer amount in nanomoles (nmol). The rule could not be simpler:

µL of buffer to add = nmol × 10

So 25 nmol → 250 µL. 38.2 nmol → 382 µL. 12.5 nmol → 125 µL. Add that volume of TE buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0) or nuclease-free water directly to the tube, vortex briefly, and let it sit at room temperature for about 10 minutes so the pellet dissolves fully. That is your 100 µM stock. Label the tube clearly with the primer name, "100 µM", and the date — future-you will be grateful.

A note on buffer choice: TE at pH 8.0 is the safer option for long-term storage because slightly acidic water can degrade oligos over months. For a working stock you will use up in weeks, nuclease-free water is perfectly fine.

Step 3: Make the 10 µM working solution

This is a straight 1:10 dilution into a fresh, labelled tube:

10 µL of your 100 µM stock
90 µL of nuclease-free water (or TE)

Mix well. You now have 100 µL of 10 µM working primer — enough for roughly a hundred PCRs. Store working stocks at −20 °C; the master stock goes to −20 °C or −80 °C for long-term keeping. Aliquoting the working stock into two or three smaller tubes is a nice touch: if one gets contaminated, you have spares.

How much primer goes into a PCR?

Most PCR protocols call for a final primer concentration of 0.1–0.5 µM. With your 10 µM working stock the maths is easy: 1 µL in a 20 µL reaction gives 0.5 µM; 1 µL in a 50 µL reaction gives 0.2 µM. Resist the urge to add extra "just to be safe" — excess primer is one of the main causes of primer dimers, which will ruin an otherwise good reaction. If your primers are well designed (see our primer design guide), the standard amounts work.

Key takeaways

Spin the tube before opening — the pellet hides in the cap.
100 µM stock: add (nmol × 10) µL of buffer. This is your master copy; freeze it and rarely touch it.
10 µM working solution: 1:10 dilution (10 µL stock + 90 µL water). Pipette daily from this.
TE buffer (pH 8.0) beats plain water for long-term storage.
More primer is not better — excess primer causes primer dimers.

Frequently Asked Questions

How do you make a 10 µM primer working solution from a 100 µM stock?

Do a 1:10 dilution: add 10 µL of the 100 µM stock to 90 µL of nuclease-free water or TE buffer. The result is a 10 µM working solution.

Should primers be stored in water or TE buffer?

TE buffer (pH 8.0) is safer for long-term storage because it guards against pH-related degradation. Nuclease-free water is fine for working stocks you will finish within weeks.

How much primer do I add to a PCR reaction?

Aim for 0.1–0.5 µM final concentration. From a 10 µM working stock, 1 µL in a 20 µL reaction gives 0.5 µM; 1 µL in a 50 µL reaction gives 0.2 µM.

New primers on the way? Check their properties first with our free Tm calculator, and confirm the GC content is in the 40–60% sweet spot before you order.