Array Advice - Hybridisation of Arrays with the Labelled Target

View tips and solutions to common problems for the hybridisation of arrays with the labelled target:

Tips

  • Take extra care when pipetting the viscous hybridisation buffer.
  • Insufficient volumes of the hybridisation mix will result in black holes on the subarrays.
  • Slowly dispense hybridisation mix to the centre of the gasket chamber. Carefully move the pipette tip around the centre to spread hybridisation mix evenly. Avoid going close to edges of gasket.
  • Monitor the temperature of the hybridisation oven using a calibrated reference thermometer. Oven temperatures can drift over time and lead to sub-optimal hybridisation conditions.
  • Avoid scratching or cracking microarray slides by ensuring that the microarray “jig”, which holds the slides together during hybridisation, is clean and rust-free before tightening.

Solutions to common problems 

Problem 1 – Black hole on array

Fig 1. Black hole on array

 Figure 1. Black hole on array

Cause: Insufficient hybridisation solution because:

  1. Incorrect volume pipetted
  2. Leakage

Solution:

  1. Ensure correct volume was pipetted
  2. Take care not to allow the hybridisation solution to touch the rubber gasket when loading
  3. Ensure the hybridisation assembly was screwed tightly before hybridisation.
  4. Check the integrity of the rubber gasket

 

Problem 2 – Non-uniform signal intensities

Fig 2. Non-uniform signal intensitiesFigure 2. Non-uniform signal intensities

Possible cause: Deformity in gasket/cracked gasket slide

Fig 3. Deformity in gasket or cracked gasket slideFigure 3. Deformity in gasket or cracked gasket slide

Solution:  Check that the gasket slide seal is intact and gasket has not cracked on disassembly.

 

Problem 3 – Bubble scarring

Fig 4. Example of bubble scarringFigure 4. Example of bubble scarring

Causes:

  1. Slide sandwich left on the bench too long before disassembly.
  2. Hybridisation oven stopped rotating during hybridisation.

Solutions:

  1. Remove slide sandwich one at a time from hybridisation oven and disassemble immediately while submerged in wash buffer 1.
  2. Ensure that the oven rotator is working prior to leaving for the day; and if possible, plug the oven into an emergency power source.

 

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