Home Analytical Chem Setting up the plate reader
Steps
  1. 1 Select the plate reader machine --:--
  2. 2 Configure wavelength and plate settings 00:20
  3. 3 Set grid area and timing parameters 00:53
  4. 4 Set up the standard curve 01:36
  5. 5 Name and configure experimental samples 02:11
  6. 6 Finalize setup and begin reading 02:51
Analytical Chem Bio-protocol Video Citable · DOI

Setting up the plate reader

DOI: 10.21769/BioProtoc.v165
Protocol
Difficulty
intermediate

Steps

1
Select the plate reader machine

Choose the Gemini XPS as the plate reader machine in Softmax Pro 6. This is the first step to initialize the instrument setup.

▶ --:--
2
Configure wavelength and plate settings

Access Settings and Kinetic Mode, then set the excitation wavelength to 544 nm and emission wavelength to 590 nm. Select 96-well Standard Opaque plate type.

▶ 00:20
3
Set grid area and timing parameters

Select the appropriate number of rows based on your sample count and configure the run time to 1 hour with 30-second intervals. Set the shake option to 5 seconds before the first read.

▶ 00:53
4
Set up the standard curve

Open Template Editor and create standard curve points in duplicate, naming each sample and entering its corresponding concentration. This establishes the reference data for analysis.

▶ 01:36
5
Name and configure experimental samples

Add and name all test samples according to your study design, specifying the dilution used for each sample. The naming will auto-increment for subsequent samples.

▶ 02:11
6
Finalize setup and begin reading

Click OK to save all template settings, then place your prepared plate into the machine and click the Read button to start the measurement process.

▶ 02:51

🚨 Failure Case Library (6) + Submit your own case

moderate
Wrong Instrument Settings for Detection Wavelength
No signal or very low signal readings from plate reader despite visible color development or expected fluorescence. Signal does not correlate with visual observations.
💡 4 · ✓ 4
moderate
Edge and Drift Effects on ELISA Plate
Wells at the plate edges show systematically higher or lower absorbance than center wells, or a gradient pattern appears across the plate, affecting data quality especially for samples in edge positions.
💡 5 · ✓ 5
moderate
Negative Fluorescence Values
Fluorescence reader displays negative values, which is physically impossible
💡 4 · ✓ 5
moderate
High CV from Poorly Maintained Plate Reader
High coefficient of variation (>15%) across the plate with no obvious pattern. Signal intensity may drift over time or show unexpected variability between identical samples.
💡 5 · ✓ 5
minor
Standard Curve OD Values Differ From Datasheet
The optical density (OD) measurement values for the standard curve vary considerably from the examples shown on the kit datasheet or protocol booklet, causing user concern about assay validity.
💡 4 · ✓ 4
minor
Plate Contamination or Optical Interference
Random high background in specific wells or patterns. Fingerprints, dust, or residue visible on plate bottom. Erratic readings not following expected pattern.
💡 4 · ✓ 5
💬 Comments coming soon