I need to run DNA template that has some ethanol left-over. I mixed it with 6x loading buffer and tried loading it on the gel but it floated up. I did a little trial adding 1:1 DNA:dye and the sample sank in the well. Now I am wondering if I use this same DNA:dye ratio with my real sample it will affect the migration rate of the DNA, specially because my ladder has a 1:5 dye:ladder ratio.
In my view, i don't think the differences in the ratio resulted in your DNA:dye not sinking to the bottom. From experience, if your DNA:dye mixture is not well mixed together before loading into a well, such a phenomenon can happen.
In order to avoid it, it will best you centrifuge briefly, the DNA:dye mixture or pipette them in and out gently before loading into well. Also recheck your well in the gel for air bubbles, which hamper sample migration.
All the best.
I guess, higher amount of the loading dye should not significantly modify DNA migration. If you are still afraid of doing it with your experimental samples, add a bit of glycerol instead of excess of loading dye (LD is just glycerol, dye and buffer) - glycerol is what makes your sample dense and forces it to sink to the well.
Probably, ethanol is what kicks your samples out of the well - ideally, get rid of it, if possible.
Additionally, make sure, you are using the correct buffer for electrophoresis. If you use concentrated stock solution (eg 10xTBE instead of 1x or 0,5x), you can observe such phenomenon.
Thanks Robert and Martin, I will do a little trial adding glycerol to my sample. Will also make sure that the dye is well mixed with the DNA before loading it.
Dye/samples not afect to much the migration rates, since the migration depends of charges applied (potential), the tickness of gel (for low size, 2%)..... Some procedures use dye after run electroforesis.
Just to share from experience I can confirm a mixture of dye:DNA changes its density according to mixing ratio. The loading dye has a lower density so when added to much it will make the samples float leaving the wells from the gel. If it is a 6x loading buffer then mix it with DNA at 1:5 ratio not 1:1. DNA migration rate (at constant applied potential and inter-electrode distance) will not change with this ratio because the limiting factor is the gel density.
I am afraid, you re not right Dorin-Mirel, loading dye has much higher density (due to glycerol or sucrose in it). DNA amples are mixed with it to sink down to the well. Addition of some dye (orange G, bromphenol blue, xylene cyanol) just helps to track your sample during elfo (and allows you to see the sample during loading, but not facilitate sinking down).
You have to mix properly the dye with the buffer and use appropriate charge
If your loading dye contains SDS it could change the migration because it will denature the DNA binding proteins. Sounds like that is not your concern, but worth keeping in mind. I don't think adding 1:1 is bad for the DNA samples. However, if your sample runs at the same rate as the dye, adding too much dye will make it very hard to see on the gel.