12 replies to this topic

[xCj425x]

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!

Edited by xCj425x, 15 July 2009 - 10:36 AM.

[mdfenko]

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?

[xCj425x]

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

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?

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!

[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.

[jiajia1987]

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

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.

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

[jah]

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:

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....halfway through the gel, and then the mixture leaks out through the top of the gel...... Is the gel of uneven thickness?"

...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

[mdfenko]

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

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.

[phage434]

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.

[xCj425x]

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.

[jiajia1987]

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

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

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.

Thanks!

[jiajia1987]

xCj425x, on Jul 17 2009, 12:07 AM, said:

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.

As for how hot is hot, it should be comfortable enough for you to hold in your hands.

What I always do is to boil about 500mL or 200mL of 1% agarose, and then keep it in a 55degcel incubator. I will then take the gel from the incubator and pour the necessary amount whenever I need to, before putting the rest back into the incubator.

[georgiadave]

I have had this happen when I try to make thin gels to save agarose.  Even if you do have a gel of even thickness there will be a larger portion around the wells...you can thank adhesion for this.  I haven't had any problems visualizing my bands on these gels either.

Try making a thicker gel or loading less.

[xCj425x]

Hi!
Yeah, so my problems are basically fixed.
It was a combination of the fact that I didn't get rid of the ethanol properly.
(After removing ethanol completely before suspending the DNA, the DNA wouldn't float out of well anymore when used in electrophoresis)

Also, georgiadave brings up a great point.
Adhesion makes it so that the gel is slightly thicker around your wells, and so when you run the gel, the DNA comes leaking out when it runs out of gel to run through, and starts pouring out of the gel itself.

Making thicker definitely helped, but as phage mentioned, putting the comb in first helps deal with the adhesion issue.
I noticed that using slightly more gel, but also assembling the comb first, resulted in a completely flat gel surface.

Thanks so much for all your help everyone!