Smears above ribosomal RNA bands
Posted 22 November 2010 - 05:53 PM
I'm trying to extract total RNA from different microalgal species.
I ran a non-denaturing 1% agarose gel and loaded 5-10 uL of each sample containing 300 - 500 ng RNA according to nanodrop measurements with 260/280 values of 2-2.1. The gel is post-gel stained with gel green, the ladder is just a normal DNA ladder and is used as guideline to compare the presence of ribosomal bands between gels.
I only get what looks like faint ribosomal bands. But what really mystifies me are the smears above what I think is the 28S band. I first thought my RNA is highly degraded, but that would mean that the fragments are shorter and travel faster through he gel. So they should be lower.
Does anybody have an idea what those strong smears could be? I will run another gel today where I add some RNase to some of the samples and maybe some samples with proteinase K, too.
Posted 28 November 2010 - 07:37 PM
In the meantime, I figured out that the smears are DNA.
Posted 08 December 2010 - 02:43 PM
before you ran your RNA in plain gel, did you heat up your RNA with glyoxal at 50dC for at least 30 min? that will show up your rRNA bands if you didn't.