DNA gel problem - (Jun/05/2008 )

I'm in desperate need of help with my DNA gel. I'm running PCR products on 2% agarose gel and I always get distinct target bands but the problem is that my marker is highly inconsistet. It shows on the heaviest of bands an dthe remaining ladder is blurry. I have ordered a new 50bp ladder but to no avail Furthermore, I have used teh same marker from labmates but I don't seem to get the distinct ladder on teh gel like they do? My gel should be alright since I can se my CR products. i red that its not always good to us eteh wells at the end of the gel?

Any advice would be welcome!

Thanks
SF

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What running buffer do you use ? How much voltage ?

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Some time loading dye also can create this problem, are you using same dye for marker and PCR product or you have prepared dye and marker mix earlier and using same in all run

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as Rawat mentionned it, they might be some degradation of your marker if the loading dye doesn't include EDTA for example (blocks the nucleases)

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I use the same loading dye for the marker and the PCR products. But the strange thing is that others in the lab using the same marker (everyone has their own individual marker tube, though) are getting their marker run well. I seem to be the only one encountering the problem.

This is how the loading dye was prepared.

0.25% bromophenol blue
0.25% xylene cyanole
30% glycerol
Water

Also, I'm running the gel at 100V for 30mins using 2% gel.

We were out of xylene cyanole and thus this was not added.

Thanks
SF

---

I use the same loading dye for the marker and the PCR products. But the strange thing is that others in the lab using the same marker (everyone has their own individual marker tube, though) are getting their marker run well. I seem to be the only one encountering the problem.

This is how the loading dye was prepared.

0.25% bromophenol blue
0.25% xylene cyanole
30% glycerol
Water

Also, I'm running the gel at 100V for 30mins using 2% gel.

We were out of xylene cyanole and thus this was not added.

Thanks
SF

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It looks to me that your marker is just getting degraded that happens to me with a 1KB ladder aliquot since then I always include EDTA in my stock marker Did you try to prepare a new gel and run side by side your maker and an aliquot of the one of one of your colleagues ? That will tell you whether your's is dgraded or if it's a gel preparation thing (which I doubt)


Jipes

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I think Jipes suggested the right thing, you have to check quality of your marker.

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The marker could be one problem but all your labmates getting good one with the same marker might mean there is nothing to worry about the marker it self.

And the 'Edge Effect' . . have a look at this thread. mdfenko had said something important.

Hope it works. . but is 'Sarwat' still around or lost?

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I ran a gel with my marker and that of the others. All the markers ran well, but a bit blurry. This time the difference was that I did not load any marker in teh first well. I read ina thread that end lanes do not run well? But why were they blurry?

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