A low reading on your Qubit fluorometer most commonly indicates that your sample's concentration is below the instrument's optimal detection range. This means the sample is likely too dilute to be accurately quantified by the fluorometer.
Understanding Low Readings in Nucleic Acid Quantification
When performing nucleic acid quantification using a Qubit fluorometer, the device measures the fluorescence emitted by a dye that binds specifically to DNA or RNA in your sample. For an accurate measurement, the amount of nucleic acid in the prepared sample must fall within the linear detection range of the assay. If the concentration is too low, the signal will be weak or non-existent, resulting in a low or "out of range" reading.
Primary Cause: Sample Out of Range
The most frequent reason for a low reading is that the sample being measured is simply not concentrated enough. This can happen if your initial stock solution is very dilute or if you have over-diluted the sample during preparation for the assay.
Solutions for Addressing Low Readings
To resolve a low reading from your Qubit fluorometer, focus on increasing the effective concentration of your nucleic acid in the sample well. Here are the key strategies:
-
Use a More Concentrated Sample:
- If possible, start with an aliquot from a more concentrated stock of your nucleic acid. This is the most straightforward way to ensure sufficient material is present for detection.
- If your current stock is already low in concentration, consider re-concentrating your sample using methods like ethanol precipitation or spin columns, if appropriate for your downstream application.
-
Reduce Sample Dilution:
- Adjust the sample preparation to include a larger volume of your undiluted sample or a smaller volume of the diluent buffer. This effectively increases the concentration of nucleic acid within the total volume measured by the fluorometer.
- Practical Example:
- If you previously diluted 10 μL of your sample in 190 μL of buffer (resulting in a 1:20 dilution), and the reading was too low, try a less dilute preparation.
- Instead, mix 20 μL of your sample with 180 μL of buffer. This creates a 1:10 dilution, effectively doubling the concentration of nucleic acid in the assay volume, thereby making it more likely to fall within the detectable range.
Here's a comparison of typical dilution adjustments:
| Initial Sample Volume | Diluent Volume | Total Volume | Effective Dilution | Impact on Concentration |
|---|---|---|---|---|
| 10 μL | 190 μL | 200 μL | 1:20 | Lower |
| 20 μL | 180 μL | 200 μL | 1:10 | Higher (doubled) |
By making these adjustments, you increase the amount of nucleic acid available to bind with the fluorescent dye, leading to a stronger signal and a quantifiable reading.
For more detailed troubleshooting and best practices for nucleic acid quantification, consult relevant technical support resources for fluorometers and nucleic acid preparation guidelines.
