I'm quite new to the thing, lousy experience with running gels.
Here's the thing.
I have some genomic DNA isolated from paraffin embedded formalin fixed tissue and I want to see how much it is degraded (formalin fixation really damages the DNA) - how bad the smear will be.
But the problem is, my DNA does not seem to want to enter the gel. It just sits in the wells.
I use 0.8% agarose and SYBRsafe dye (I add it to the agarose before pouring the gel) and 1x TAE, the run goes for 3.5 hrs at 60 V (4 V/cm). The running conditions are what puzzles me the most.
I mix approx 1 uL of loading dye (sucrose, TAE, xylene cianol and bromphenol blue) with 4 uL of the DNA. I cannot tell you the precise amount (varies greatly between samples), but there definetly is DNA in there (concentrations measured with Qubit and all fine)
After visualization, there is only some pale fluorescence arount the wells, but no bands/smears. And even my ladder does not show (I'd be glad to attach the picture of this mess, but the attachment manager says that's not a valid image type). Also, the paleness disturbs me a bit, since SYBR is supposed to shine more, as it is much more sensitive than EtBr. But at the moment, that's the minor problem.
Anyway. I first thought it might be something with the buffer, but I see bubbles and the indicator dyes migrate perfectly, just where they are supposed to migrate.
Maybe I'm doing some very obvious error, but this are my first attempts (I did some electrophoresis of PCR products and a RFLP run once during undergrad lab practice). I guess I'm all wrong about the running conditions, but I really don't feel like trying them out over and over and wasting time, agarose and SYBR.
Could someone please help?