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Troubleshooting

Weak or no signal

Possible Cause Possible Solution
Did not add DTT/Tween® 20 to binding reaction Add 1 uL of 25 nM DTT/2.5% Tween 20 to binding reaction
Not enough IRDye® labeled DNA used Increase amount of IRDye labeled DNA added to the reaction
Target DNA degraded Verify integrity of DNA
Imaged in the wrong channel of the Odyssey When using IRDye 700-labeled DNA, turn on the 700 nm laser.

No shift bands detected or weak signal

Possible Cause Possible Solution
Auto Sensitivity selected in Odyssey Software Change sensitivity setting to manual and adjust Sensitivity manually. Use Image Studio software and the Auto Scan mode.
Scanned gel with intensity too low Increase intensity parameter in Odyssey software to 8 and scan again. Use Image Studio software and the Auto Scan mode.
Incorrect focus offset Adjust the Focus Offset in the Odyssey software or Image Studio software to equal the thickness of the glass plate plus half the thickness of the gel, and scan again.
DNA:protein complex disrupted due to heat or vortexing Run gel with cooled buffer. Do not vortex binding reaction.
Not enough extract Add more extract to reaction.
Degraded extract Minimize freeze/thaw cycles. Use protease inhibitors.
System not fully optimized Use additives in the kit (Odyssey EMSA Buffer Kit, P/N 829-07910) to determine their effects on binding efficiency.

Spots or speckling

Possible Cause Possible Solution
Contamination on glass surfaces Clean glass gel plates and the Odyssey scanning surface with isopropanol


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