Most likely the problem is with your loading.  Unlike in an SDS-PAGE gel there is very little stacking effect in a TBE-PAGE gel and so if the DNA solution is not loaded very carefully into the bottom of the well or if there are irregularities in the shape of the wells then these irregularities will show up as distorted bands.  After pouring the gel and pulling the comb immediately wash the wells with a little 0.25X TBE to ensure any slight bits of unpolymerized acrylamide are cleaned out and examine the wells to ensure the they are uniform without defects. I can see from the spread-out shape of the bands that your wells were not completely flat-bottomed and had a slope back-to-front, hence when looking through the gel thickness the bands seems smeared or doubled as in the ladder. I also agree that you should try the above suggestion of loading less DNA, in the agarose gel there is some streaking of DNA in front and behind the major bands, acrylamide gels are much more sensitive so you are probably just picking up some more of this material.  Also, as a final point, it is important in my experience when running TBE gels to assemble the gel apparatus and pre-run the gel for about 5 - 10 min before loading any DNA in it. This ensures that any slight difference in the concentration of salts between the gel and the running buffer is run into the gel before the DNA is loaded and thus won't cause irregular migration of the bands.  

Hello Tung,

which staining procedure did you use for PAGE? Could it simply be that your staining procedure for PAGE is more sensitive than your EtBr staining of the agarose gel?

If you tried to use denaturating PAGE with a denaturating agent such as urea, your mispreparation may degrate your DNA during electrophresis. More over if you used different type of loading dyes , it could degrate your DNA too. It is definately a degredation problem during electropharesis or sample preparetion.

However, if you check your marker bands, they are not smeared in PAGE too. But your DNA in agarose jel has a very low background smear in agarose too. What I mean is that, your DNA can be degredated in agarose jel too but agarose is not enough efficient to see degredated fragments as good as PAGE. So please try agarose with a lower density but more stain, maybe you can see fragments in that way.

Have you tried loading 3-5X less of those DNAs; that is a lot of signal for only one band? There could also be some heterogeneity in your DNA.

Most likely the problem is with your loading.  Unlike in an SDS-PAGE gel there is very little stacking effect in a TBE-PAGE gel and so if the DNA solution is not loaded very carefully into the bottom of the well or if there are irregularities in the shape of the wells then these irregularities will show up as distorted bands.  After pouring the gel and pulling the comb immediately wash the wells with a little 0.25X TBE to ensure any slight bits of unpolymerized acrylamide are cleaned out and examine the wells to ensure the they are uniform without defects. I can see from the spread-out shape of the bands that your wells were not completely flat-bottomed and had a slope back-to-front, hence when looking through the gel thickness the bands seems smeared or doubled as in the ladder. I also agree that you should try the above suggestion of loading less DNA, in the agarose gel there is some streaking of DNA in front and behind the major bands, acrylamide gels are much more sensitive so you are probably just picking up some more of this material.  Also, as a final point, it is important in my experience when running TBE gels to assemble the gel apparatus and pre-run the gel for about 5 - 10 min before loading any DNA in it. This ensures that any slight difference in the concentration of salts between the gel and the running buffer is run into the gel before the DNA is loaded and thus won't cause irregular migration of the bands.  

The sensitivity of the DNA-PAGE is much higher than the agarose gel. Try another gel by loading lesser amount of sample and possibly it resolves your problem. Since the smearing is at higher molecular weight DNA; there is no issue of sample degradation. During the second run start electrophoresis at lower voltage and increase it when the sample is entered into the gel it will increase the stacking effect.

What's in your DNA loading dye? If it is glycerol, try to replace it with ficoll-400.

The capacity and resolution of PAGE gel and Agarose gel are different. If you run your DNA on  2% and  0.7%  agarose gel, you will see less or more smear on the gel at particular size fragments too. Same apply to if you run 4% and 8% of the PAGE gel. Your have the smear(mixed DNA fragments) in your DNA pool, depends on how you run your DNA.

Hi, thank you very much for all suggestions.

I tried a new water (HPLC grade) for gel running but saw no difference. So this is not a problem with my DI-water.

I took more caution in casting the PAGE gel and loading the samples. As can be seen from the sharp look of the ladder, any distortion at the bottom of the wells can be excluded in this new gel.

The fact that I saw additional weak bands, not just smeared band, may suggest that there is some heterogeneity in my DNA samples.

Thanks.  

How do you cast your gels in terms of amount of ingredients? The protocol I'm using makes the acrylamide shrink significantly and the wells never come out perfect.

I know there is always a percentage of shrinkage but in the TBE-PAGE gels it's very obvious compared to SDS-PAGE gels where I had wells come out very flat.

My typical recipe for 10-mL of 2% native PAGE gel (29:1 ratio) is:

7.265 mL diH20

625 uL glycerol 80%

1 mL TBE 5X

1 mL 40% Acryl-bis 29:1 solution

100 uL APS 10% w/v

10 uL TEMED

I did not observe any shrinkage upon complete polymerization.