I ran these 15% gels for 90 minutes under 200 volts ( gel ran too slow), ladder looked very condense on the top and the dye front was somehow diffused at the bottom of the gel. I am sure about the gel recipe and samples. Does anyone know what causes this issue? Is it the use of excessive APS or use of old acrylamide/tris buffer?
Some people use constant current for SDS-PAGE, but most of the time I use constant voltage for my minigel. Try to run through the stacking gel at lower volts (50 or 60 V) Then, the resolving gel can be run at 100 V or 120 V. People use 200 V if in a rush, but this can give more diffuse bands. Try to reduce your voltage from 200V. Too much heat can partially melt your gel. Check the current too. If the current in a minigel is getting significantly more than 50 mA, you should reduce the voltage.
Good luck...
change the buffers you used . tank , gel and sample buffers .prepare them freshly.
As Dr Sahadevan says, 200 V is too much voltage, I perform SDS-PAGE (16%) applying 150 V (corresponding initially with 40 mA).
Jasim Al-saadi
You were right, I changed all the reagents and now everything works fine. Thanks everyone for suggestions.
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