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Concentration measurements

Nanodrop

Overview

Manual

  • Detection limits for dsDNA/RNA: 2-10,000 ng/uL
  • Light is passed through the sample (190-850 nm)
  • DNA and RNA absorb UV light at 260 nm
  • Baseline correction is applied to offset light scattering in a particular sample. It is measured at 340 nm where no sample-related absorption is expected.
  • Extraction reagents such as guanidine, phenol, and EDTA contribute absorbance between 230 nm and 280 nm and will affect measurement results if present in samples. |400

Procedure

  1. Wet a small piece of tissue with Milli-Q water and clean the sensors on the device and on the lid
    • Do not apply too much water as liquid can get into the device and cause damage.
  2. Pour 2 uL of the medium in which your sample is suspended (e.g., elution buffer or water) on a sensor.
  3. Select the appropriate channel for measurements.
  4. Run blanking (click on a water drop symbol).
  5. If successful, open the lid, wipe off the sensors (don't forget the sensors on the lid!).
  6. Pour 2 uL of your sample on a sensor.
  7. Hit run (click on a triangle symbol).
  8. Results:
    • Amount in ng/uL
    • A260/A230: DNA to salt ratio should be around 1.8-2.2.
    • A260/A280: DNA to protein ratio should be ~1.8, RNA to protein ratio should be ~2
    • An orange ! is shown if a ratio is out of bounds.
    • An orange A appears if certain contaminants were identified. Click in the icon to see the analysis.

Qubit

Specifications

  • Highly selective for dsDNA over RNA
  • Dye choice depends on the expected range of concentration:

Procedure

  1. Prepare n + 2 lo-bind .5 mL Eppendorfs, where n is the number of samples and 2 are the two standards.
    • The standards and samples are stable for 3 hours if protected from light.
  2. Make a master mix with 199 uL x (n + 2) and (n + 2) uL of dye.
  3. Add 190 uL to the two tubes for standards + 10 uL of Standard 1 / 10 uL Standard 2.
  4. Add 195 uL to the remaining tubes + 5 uL of your sample.
  5. Incubate for 2 min to make sure your sample and the standards are room temperature.
  6. Measure your standards in the Qubit device.
  7. Set the sample size to 5 uL in the Qubit interface and measure each sample.