Bromophenol blue question

[DacotahM]

My bromophenol blue dye will not migrate across a gel reliably. Sometimes it seems to work properly but other times it will sit in the wells and refuse to move. The ladder and Gotaq Green commercial dye work fine on the same gel.

The recipe I use is

30mL glycerol
70mL ddH20
0.025g bromophenol blue

which I dilute to 1:6 with 1x TBE.

It will randomly work perfectly or fail completely to migrate into the gel. I have tried spinning it down and using the supernatant but that is still unreliable.

[butters]

Is the loading dye you make very blue?

I found it is ok to reduce bromophenol blue. Important regarding your question. Yes about 30% glycerol is 6x, so just add one part of you solution with five part PCR or DNA (yes you can add less).

If you dilute it with 1:6 buffer then your loading may not be able to bring all DNA to sit on bottom of well.

hmmmm, when you run, did you see bubble formation on anode or cathode? there are known cases where power pack was not connected/connected on the wrong power pack... check that out. If there are bubble mean voltage are apply.

and what voltage did you use for what length of gel?