agarose-gel artefact - (Feb/13/2006 )

Everytime I check the Lightcycler products on an agarose-gel, I get a strange band, apparently weightier than the specific product. It's very light against the background and can only be seen with good eyes or after adding contrast to the picture with Photoshop. It always appears in addition to a PCR product. But it doesn't matter, if the PCR starts from genomic or plasmid DNA. It appears with plasmid-DNA, that starts from only one copy and with plasmid DNA, that starts with 10^7 copies. The melting curves say, that there is only the specific product. Now, it happened, that this shadow band is much stronger, than everytime before. There is no hint from the melting curve, and it has the same shape as the specific product.
Can anybody confirm, that this is an artefact? I'm using Roche SYBR-green mastermix. Perhaps it acts as a glue, when I load PCR-product (8µl] on the gel, so that a small fraction of PCR-product runs delayed through the gel.

thanks for your answers

westenmax

hi
i don't think the PCR product would migratesat lower rate, since in that case the lower band would be an artefact too.
I would suggest to heat the sample brifly 5' 60° before lodaing in order to remove the possible secondary structures generated by that.
For chacking if it's a loading problem, you can gel purify the band of interest and re do a PCR. If it's a problem of migration, you'll see 2 bands. If not, you'll need to go to a contamination hypothesis