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:
- 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
Figure 1. Black hole on array
Cause: Insufficient hybridisation solution because:
- Incorrect volume pipetted
- Ensure correct volume was pipetted
- Take care not to allow the hybridisation solution to touch the rubber gasket when loading
- Ensure the hybridisation assembly was screwed tightly before hybridisation.
- Check the integrity of the rubber gasket
Problem 2 – Non-uniform signal intensities
Figure 2. Non-uniform signal intensities
Possible cause: Deformity in gasket/cracked gasket slide
Figure 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
Figure 4. Example of bubble scarring
- Slide sandwich left on the bench too long before disassembly.
- Hybridisation oven stopped rotating during hybridisation.
- Remove slide sandwich one at a time from hybridisation oven and disassemble immediately while submerged in wash buffer 1.
- 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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