| Possible Cause | Possible Solution |
|---|---|
| Did not add DTT/Tween® 20 to binding reaction | Add 1 µL 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® Imager | When using IRDye 700-labeled DNA, turn on the 700 nm laser. |
| Possible Cause | Possible Solution |
|---|---|
| Used incorrect scan intensity setting | Scan the gel again using a Scan Intensity of 8 in the Odyssey Application software, or use the AutoScan mode in the Image Studio™ software |
| 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. |
| EMSA binding reaction not fully optimized | Use the additives in the Odyssey EMSA Buffer Kit (P/N 829-07910) to optimize the binding reaction conditions. |
| Possible Cause | Possible Solution |
|---|---|
| Contamination on glass surfaces | Clean glass gel plates and the Odyssey scanning surface with isopropanol |