It took much longer for me to get a round to testing out my gel electrophoresis equipment than I thought it would. For now I have merely got it to work. Next I will try and fine tune it to increase the quality of the gels. More on that below.
This isn’t the most informative post but I was kind of frustrated by a lack of information when I was troubleshooting so I figured I would throw some data out and hope it helps someone.
Note: The cameras on my phone and iPad both captured more wavelengths of light than I could see, so these images look worse than the gel actually is.
- GEL = 1.5% food-grade agar-agar gel (not agarose)
- DNA LADDER= New England Biolabs 2-log DNA ladder
- STAIN = GelRed Stain (Vendor; Biotium) (Approx. Equiv. to Ethidium Bromide, except safe). Stain was used in the precast gel (1x) context.
- TRANSILLUMINATOR = Fotophoresis I (Fotodyne)
- BUFFER = TAE (MB grade reagents)
The image above is what the first two gels looked like – no fluorescence at all. I still do not know for sure why they failed but I narrowed it down to either the composition of the DNA ladder or the staining method.
My set-up worked when I used GelRed in the molten agar-agar and composed the DNA ladder per the manufacturers instructions. I used 5-10uL of ladder and 1-2uL of loading buffer on the failed gels whereas I used 1uL of ladder, 4uL H2O, and 1uL loading buffer on the successful gel. During the failed gels I tried to use the 3x post-electrophoresis stain procedure with staining times between 0.5-1.5 hours – all with no luck. Obviously changing two variables at once confounds the results – but at least I have a baseline now.
This was the gel box I built and used. You can read my instructions for how to build it here.
This is ~90% of my apartment lab.
My next goal is to work out how to fine tune the procedure. I am going to compare the gel quality in cases where I use reagent grade vs. industrial and food grade chemicals. I am hoping that borax (sodium tetraborate) and roach poison (boric acid) buffer will work as good as its reagent grade cousin.