Hello everyone,

I’m currently running a Comet Assay and have encountered an issue where the voltage during electrophoresis can’t go above 10V, even though I’ve set the current to 500 mA and power to 50 W. I’ve double-checked the equipment, and it works fine when using TBE buffer, but when I switch to the Comet Assay buffer (300 mM NaOH, 1 mM Na₂EDTA), the problem persists.

Buffer Preparation:

I’ve tried preparing the buffer both from stock solutions and directly from raw materials. Here are the details:

1. Preparation Using Stock Solutions for 400 mL:

• 10 M NaOH stock solution:

• I used 12 mL of 10 M NaOH (prepared by dissolving 40 g of NaOH in 100 mL ddH₂O).

• 200 mM Na₂EDTA stock solution:

• I used 2 mL of 200 mM Na₂EDTA (prepared by dissolving 7.44 g of Na₂EDTA in 100 mL ddH₂O).

• The final volume was brought to 400 mL using 386 mL ddH₂O.

2. Preparation Without Stock Solutions for 400 mL:

• NaOH:

• I directly weighed 4.8 g of NaOH (40 g/mol) to get 300 mM for 400 mL.

• Na₂EDTA:

• I directly weighed 0.149 g of Na₂EDTA (372.24 g/mol) to get 1 mM for 400 mL.

• Dissolved the solids in 350 mL of ddH₂O and brought the volume to 400 mL.

Troubleshooting:

1. I’ve confirmed that both NaOH and Na₂EDTA are fully dissolved.

2. The pH of the buffer is >13.

3. I’ve checked the equipment, and it works fine with the TBE buffer, but the voltage remains stuck at 10V with the Comet Assay buffer.

Does anyone have suggestions, or has anyone encountered this problem before with Comet Assay electrophoresis buffer? Could it be a problem with the buffer conductivity or other factors?

Any advice would be greatly appreciated!