Is there a commonly protocol for the quantification of DNA with a platereader?
Since it works the same way as a UV/VIS spectrometre, platereaders should be usable for quantification of DNA. Is there a common ground regarding the parameters? volumes, duplex or triplex sample preparation for example?
Best regards,
Mathis
Sample Preparation:
Dilution: DNA samples are often diluted in a suitable buffer (like TE buffer or water) to bring them within the optimal detection range of the plate reader.
Volume: The volume of each sample in the wells should be consistent and within the recommended range for the plate reader. Typical volumes range from 100 to 200 µL per well, but this can vary depending on the plate type and reader specifications.
Plate Type:
Material: Plates made from UV-transparent materials, like quartz or UV-transparent plastic, are used to allow accurate measurements at 260 nm.
Well Count: 96-well plates are common, but other formats (like 384-well plates) can be used depending on the throughput needs and the capabilities of the plate reader.
Measurement Parameters:
Wavelength: Measure absorbance at 260 nm for DNA quantification. Additionally, measurements at 280 nm are often taken to assess protein contamination (as proteins absorb strongly at this wavelength). The ratio of absorbance at 260/280 nm provides an estimate of DNA purity.
Reference Wavelength: Some protocols use a reference wavelength (like 320 nm) to account for background absorbance.
Standards and Controls:
Standard Curve: Use DNA standards of known concentrations to create a standard curve. This is critical for accurate quantification.
Blank: Include a blank (buffer without DNA) to calibrate the zero point for absorbance readings.
Data Analysis:
Calculation of Concentration: Use the absorbance values and the standard curve to calculate the DNA concentration in your samples.
Purity Assessment: Evaluate the purity of the DNA by looking at the 260/280 nm ratio. Pure DNA typically has a ratio of ~1.8.
Duplex or Triplex Sample Preparation:
If you're comparing samples or need to account for variables like different buffers, it might be useful to prepare duplicates or triplicates of each sample to ensure reliability and reproducibility of your measurements
- Badges
- Science topic
- Similar topics
- Philosophy
- Esthetics
- Interpretation