Oligo (Primer) Resuspension

The First Step to Using Synthetic DNA.

What is Oligo (Primer) Resuspension?

Oligonucleotide (or 'oligo') resuspension is the process of dissolving a dehydrated, synthetic piece of DNA (like a PCR primer) into a liquid to create a usable stock solution of a specific concentration.

Companies that synthesize DNA typically ship it in a stable, lyophilized (freeze-dried) state as a small pellet at the bottom of a tube. Before it can be used in an experiment, it must be carefully resuspended in a buffer or nuclease-free water.

The goal is to create a concentrated, stable stock solution (e.g., 100 µM) that can be easily diluted later to a final working concentration for experiments like PCR.

Example: The small, often invisible pellet of DNA must be dissolved in a precise volume of liquid to achieve the desired stock concentration.

The Key Information: Moles on the Tube Label

The most critical piece of information provided by the synthesis company is the total amount of oligo in the tube, usually given in nanomoles (nmol).

This value tells you the exact number of moles of DNA you have. With this information, you can calculate the exact volume of liquid needed to reach any desired molar concentration.

Example:A tube label might read '24.7 nmol'. This is your starting point for all resuspension calculations.

The Core Formula: Moles = Concentration × Volume

The calculation is based on the fundamental relationship between moles, concentration (molarity), and volume.

Amount (moles) = Molarity (mol/L) × Volume (L)

For resuspension, we rearrange this to solve for the volume of liquid we need to add:

Volume to Add (L) = Amount (mol) / Desired Molarity (mol/L)

It is critical to be mindful of units! Lab work is often done in microliters (µL) and micromolar (µM), so conversions are almost always necessary.

Example:This simple formula is all that is needed to accurately create a stock solution of any desired concentration.

A Practical Calculation Example

Let's walk through a common scenario:

Goal: Create a 100 µM stock solution from a tube containing 24.7 nmol of a DNA primer.

Step 1: Convert Units to be Consistent. Let's convert everything to moles and Liters. Amount = 24.7 nmol = 24.7 x 10⁻⁹ mol. Desired Concentration = 100 µM = 100 x 10⁻⁶ mol/L.

Step 2: Calculate Volume in Liters. Volume (L) = Amount (mol) / Molarity (mol/L) = (24.7 x 10⁻⁹) / (100 x 10⁻⁶) = 0.000247 L.

Step 3: Convert to a Practical Unit (Microliters). Volume (µL) = 0.000247 L * 1,000,000 µL/L = 247 µL.

Result: To create a 100 µM stock, you would add 247 µL of buffer or nuclease-free water to the tube.

Example:**Shortcut:** A very useful lab trick is to note that the volume in µL required to make a 100 µM solution is simply the number of nanomoles multiplied by 10. (24.7 nmol * 10 = 247 µL).

Real-World Application: Preparing for PCR

Proper oligo resuspension is the first step in countless molecular biology workflows.

PCR Primer Stocks: PCR experiments are highly sensitive to primer concentration. Creating an accurate 100 µM stock solution is standard practice. This stock is then used to make a more dilute (e.g., 10 µM) working solution, which is then added to the final PCR reaction.

DNA Probes: Fluorescently labeled DNA oligos used as probes in techniques like qPCR or FISH (Fluorescence In Situ Hybridization) must be resuspended to a precise concentration to ensure consistent and quantifiable results.

DNA Sequencing: Primers used for Sanger sequencing must be at the correct concentration to ensure the reaction works correctly.

Example:An error in the initial resuspension of a primer stock will carry through all subsequent dilutions and can be a major cause of failed or inconsistent experiments.

Key Summary

**Oligo Resuspension** is dissolving a dry DNA pellet to create a concentrated stock solution.
The key calculation is based on the amount of DNA in **nanomoles (nmol)** provided by the manufacturer.
The formula is **Volume = Amount / Concentration**. A common stock concentration is 100 µM.
A useful shortcut: to make a 100 µM stock, add a volume in **µL** that is **10 times** the number of **nmol**.
Proper resuspension is critical for the accuracy and reproducibility of downstream experiments like PCR.

Practice Problems

A tube of a DNA primer arrives with 35.2 nmol of oligo. What volume of water (in µL) do you need to add to make a 100 µM stock solution?

Use the shortcut: Volume (µL) = nmol * 10.

Solution: Volume = 35.2 * 10 = 352 µL.

You have a tube containing 50 nmol of a primer. You want to make a 20 µM working solution directly. What volume of buffer (in µL) do you need to add?

1. Convert units: 50 nmol = 50 x 10⁻⁹ mol; 20 µM = 20 x 10⁻⁶ mol/L. 2. Calculate Volume (L) = Amount / Molarity. 3. Convert L to µL.

Solution: Volume (L) = (50 x 10⁻⁹) / (20 x 10⁻⁶) = 2.5 x 10⁻³ L. Volume (µL) = 2.5 x 10⁻³ L * 1,000,000 µL/L = 2500 µL, or 2.5 mL.

Frequently Asked Questions

What should I resuspend my oligos in?

Oligos can be resuspended in sterile, nuclease-free water. However, for long-term storage, it's better to use a low-salt buffer like TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0). The buffer helps to maintain a stable pH and the EDTA chelates divalent cations, which can be cofactors for DNA-degrading enzymes (nucleases).

How do I make sure the oligo is fully dissolved?

After adding the liquid, you should let the tube sit for a few minutes. Then, vortex the tube for 15-30 seconds to mix thoroughly. Finally, perform a quick spin in a microcentrifuge to bring all the liquid down to the bottom of the tube.

How should I store my resuspended primer stock?

For long-term storage, primer stock solutions are best stored frozen at -20°C. For daily use, it's common to keep a small aliquot (a 'working stock') in the refrigerator at 4°C to avoid repeated freeze-thaw cycles, which can degrade the DNA over time.

Oligo Primer Resuspension

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