Agarose: DNA/dye mixture doesn't stay in the gel? - Thanks for any and all help received! =] (Jul/15/2009 )

Hi everyone!
I had a quick question regarding agarose gel electrophoresis.
So I have my gel made, and I have my DNA/dye mixture (I usually use a ratio of 1uL 5x loading dye: 4uL DNA), and then I load the mixture into the wells. So far so good.
I start running the gel. So far so good, then maybe five minutes in, the dye starts leaking out of the well/gel. At least some of the mixture is still traveling as usual through the gel through, because in the end, I still get visible bands, but it makes me wonder just how much DNA is randomly leaking out.
Sometimes the DNA mixture doesn't leak out of the well, but rather it is running just fine, halfway through the gel, and then the mixture leaks out through the top of the gel.

My question is basically, "What am I doing wrong? Is the loading dye concentration too low? Is the gel of uneven thickness?" (Okay that's like three questions)
I was just wondering if anyone had any similar issues, and if he or she successfully fixed the problem.
Thanks for reading!

is there a possibility of any ethanol left in your sample? that would make your sample less dense than the buffer.

is there glycerol in your sample buffer? do you make it yourself or do you purchase it?

are you using he right ratio?

mdfenko on Jul 15 2009, 02:38 PM said:

Hm.. could be an ethanol issue.
Perhaps when isolating the DNA, I need to make sure the ethanol is completely dried out before resuspending DNA.

If by the sample buffer you mean the TAE buffer we use to run the gels in, there should be no glycerol.
We make it ourselves. We as in, all I really do is take the 25x TAE buffer and dilute it down to .50x, and then use it in my gels.

Sorry, just to clarify, which ratio are you referring to? If you mean the loading dye/dna ratio, I'm using a 1:4 ratio that I've been using since I was hired at the beginning of the summer.
The only difference is that before I never had issues like this, but now I do.

It might be because I am getting sloppy with the ethanol.
Thanks for your help mdfenko!

i agree, it's probably the ethanol.

the sample buffer to which i was referring is the loading dye. many of the commercial loading dyes are 5-6x so your usage looks okay.

mdfenko on Jul 16 2009, 02:51 AM said:

I am sorry if this sounds dumb, but I would like to know why ethanol would make the sample leak out of the gel?

Not to contradict the venerable mdfenko, but if the dye/sample does not float out of the well before applying the current, I'm not so sure that ethanol is your problem here. If I understand correctly, paraphrasing elements of your query....

xCj425x on Jul 15 2009, 02:24 PM said:

...the sample does not float out of the well, but rather comes out along the lane as the sample migrates through the gel - Correct? If so, then it sounds your gel may not be of even thickness along the length of the gel....is it perhaps thicker at the wells than at the opposite end? Make sure that the gel tray is level when you pour the gel. Many casting stands come with a bubble-level, and you can adjust the tray with thumb-screws to restore proper pitch. In less-well equipped labs I've had to improvise a bit, but you want to be as close to level as possible.

Another possibility, might be that your voltage is too high, causing the gel to warm, soften and release the dye. This happened to me once when I was running a plasmid digest through low-melt agarose and forgot that TAE buffer which requires lower voltages (7-10 V/cm) than the 'Fast' borax buffer I use for short RT-PCR products (12-15V/cm).

Of course, it never hurts to ensure that the EtOH is evaporated

jiajia1987 on Jul 15 2009, 09:20 PM said:

ethanol will make your sample less dense than the surrounding buffer.

jah has a point. i was only considering the sample floating out of the well.

I have had a situation where very hot agar produces gels with small holes at the bottom of wells, which then leak the DNA sample. You might want to check for this, and allow the agar to cool a bit before pouring it into the gel tray. Also, assemble the comb before pouring the hot agar.

Yeah, I was wondering about that too.
I'll make sure about the ethanol next time I extract DNA.

But the gel thickness issue definitely also makes sense regarding why the mixture would float out of the gel itself, instead of just the wells. I know that the apparatus is level, but the middle of my gel always is lower than the wells itself.

Also, I could definitely be using overheated gel. The problem lies in the fact that I don't exactly know how hot is "too hot"
I always just assumed that as long as it's melted, it's okay, so I usually end up heating it up to when I know for sure it's hot enough. Whoops.

Thanks so much for all the responses!
I'll post again after applying the fixes mentioned above.

mdfenko on Jul 16 2009, 09:25 PM said:

Thanks!