To dilute a lyophilized primer with water to create a 100 µM stock solution, you need to determine the amount of water to add based on the number of nanomoles (nmol) of primer in the tube. Here's a step-by-step guide:
Determining Water Volume for Primer Dilution
The key to diluting a primer is calculating the correct water volume to achieve the desired concentration. The reference specifies a simple calculation:
- Multiply the number of nmol of primer by 10. This will give you the required volume in microliters (µl) to achieve a 100 µM stock concentration.
Example
Let's say your primer tube contains 38.2 nmol of primer.
- Calculation: Multiply 38.2 nmol by 10.
- 38.2 nmol * 10 = 382 µl
- Result: You should add 382 µl of water to the primer tube to create a 100 µM primer stock solution.
How to Perform the Dilution
- Obtain Lyophilized Primer: Get the tube of lyophilized primer from the manufacturer.
- Calculate Water Volume: Based on the nmol listed on the primer tube, calculate the volume of water needed using the method described above.
- Add Water: Using a micropipette, carefully add the calculated volume of sterile, nuclease-free water to the primer tube.
- Mix Thoroughly: Gently mix the solution by pipetting up and down several times. Avoid creating bubbles.
- Store Correctly: Store your newly diluted primer stock at the recommended temperature for optimal results. Usually -20°C or -80°C.
Key Concepts
| Concept | Description |
|---|---|
| Lyophilized Primer | Primer provided in dried form (typically from the manufacturer) |
| Nmol (Nanomole) | A unit of measurement for the amount of a substance |
| µl (Microliter) | A unit of volume measurement |
| 100 µM Stock | A stock solution of the primer at 100 micromolar concentration |
| Sterile Water | Water that has been purified and free from any possible microbial contaminants |
Important Considerations
- Water Quality: Always use sterile, nuclease-free water for primer dilution. This will prevent contamination and preserve the integrity of your primer.
- Pipetting Accuracy: Accurate pipetting is crucial for preparing precise stock solutions. Use calibrated micropipettes.
- Storage: Ensure you label your diluted primer stock clearly, indicating the date of preparation and concentration.
- Working Solutions: Usually, the 100 µM stock solution is further diluted to create a working solution for your specific experiment.
By following these steps, you can effectively and correctly dilute your primer with water for use in your experiments.
