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problems with electrophoresis run - (Oct/27/2008 )

hi guys!

Lately, I had several bad results with electrophoresis run.

I always used 1.5% agarose gels, 80 V and added Sybr Green directly to ladders and samples before loading. I can't use Ethidium because of safety issues.
I began to find something like a big bright band at the end of the run of both ladders and samples, as you can see in the picture. In that case, you can see the ladder, 8 negative samples, the negative control (water) and the positive control (500 bp band).
I thought it could depend on the staining procedure, so I began to perform post-run staining with Sybr Gold. I've tryed from 10' to 30'-long stainings of the gels without improvements.
It can' depend on the loading dye (orange/blue) because it's added to ladder but not to the samples (I use a PCR master mix already containing dyes), and it can't be primer dimers, because in that case it would not be visible in the ladder run. In addition, I found this fake band in several PCRs, so it's not specific of this particular reaction or samples.
It seems to be less intense in the negative control (water, penultimate lane) and in the positive samples.

What do you think it is? What can I do to avoid it?
Thanks for any suggestions!



It looks to me like a contamination of some sort, I would say that you have contaminated more than one tube from your PCR or loading dye and the ladder tube.


Hi bob1 and thanks for your help!
What do you exactly mean with contamination? What kind of contamination? Do you think I've carried some DNA over the ladder and the samples tubes?
They have nothing in common: loading dye, water and so on are added in different moments from different stocks. even the tubes I used are kept in different places.
Moreover I've faced the same problem with both Sybr Green and Sybr Gold.
Do you think the problem can rely on TBE stock???