DNA does not migrate on gel

[Enne815]

I have tried running many samples of different insert sizes and constructs on a 0.8% agarose gel. The ladder migrates but the DNA appears to migrate higher than the ladder.

I have tried:

New 1XTAE
Different rig
Different power supply
Various DNA from different stocks
New Agarose

Even changing all of these factors no DNA will migrate correctly.

Does anyone have any suggestions?

Thanks!

[Andriy]

Wow, that's a tough one...

What about loading buffer?

Do you add the same loading buffer to all of your samples? Make sure it's prepared with TAE and not with something else

Andriy

[Andriy]

What about the loading dye? Are you using the same loading dye for your ladder and for all of your DNA samples? Make sure it is made with TAE and not something else

[Aga]

It seems that it's really the problem with your loading dye. Check whether you need more amount of it than you use. I has similar problem with my loading dye when I noticed that the marker was fine but my samples were not loaded into the well and seemed to 'float'.
I just changed the loading dye and everything was fine. I occured that I prepared my loading dye with too little amount of glycerol.

[ramnivas]

I think it may be the problem either due to buffer of loading dye. I think you can try 1X TBE buffer in place of TAE.

[Aga]

You need to be make sure if:
- you DNA ladder is of correct size - are your samples within the range of the ladder you use?
- buffer doesn't form a layer above your gel more than 1-2mm thick - if you add too much buffer your DNA will migrate through the buffer and not through the gel. Hovewer you say your ladders migrate well, so check your loading dye.

And if you try to apply any changes - give your feedback - it may be informative for us all.

[Archonster]

sample float may be caused by the problem of your loading dye or dilution problems.loading dye contains sucrose which can make the sample to sedimate more quickly

[pigfarmer]

Have you ran samples that you know will run well on the gel? Perhaps if not the loading dye you have ethanol left in you sample?

[Enne815]

So, I finally determined the problem. It appears the filters on the department's Milli-Q water dispenser were not changed in 1 and 1/2 years. Once the filters were changed, my gels worked fine. A simple solution, but for me, not the most obvious. So my advice is: when all else fails, be weary of the water.

Thanks you for all of your suggestions!

[thebetafemale]

Interesting. I'm having a similar problem, only my ladder won't migrate either! I've been trying to do this gel isolation for days now and each time I run the (50 microliter) PCR, load the samples onto the gel, run it at 51 constant volts for 2.5 hours, and they don't migrate! The ladder just looks like a smear near the well and there appears to be bands, but they are just a few millimeters below the well. I'm using a different loading dye for the ladder than I am for my samples, and the dye migrates just fine. The DNA just won't. I know it's a 1% gel because I mix it up fresh each time and measure out the agarose exactly. What the hell am I doing wrong????