I'm labeling with biotin-dUTP the restriction-digested MAP7 DNA that I want to attach to my streptavidin-coated beads. I wasn't sure what length range of DNA fragments would be best, so I've cut some with XhoI (mean length ~12 kb, ~4 µ) and some with EcoRI (mean length ~6 kb, ~ 2 µ).
After I've purified the biotin-tagged DNA (must get rid of ALL of the free biotin), I'll test its ability to bind to the beads by mixing some DNA with some beads and running the mix in an agarose gel. DNA that's bound to the beads should stay in the well rather than entering the gel, because the beads will be bigger than the gel mesh size. Well, the beads will be 1 µ or 2 µ in diameter (I'll try both, one with the XhoI-cut DNA and one with the EcoRI-cut DNA), and I'm quite sure that's much bigger than the mesh size of a 0.8% agarose gel. As controls I'll use the same DNA digests without the biotin tagging.
I'm also making more B. subtilis DNA for my transformation tests, because the first prep didn't have much DNA in it. I'll pool both preps and repurify by another round of phenol extraction and spooling.
Tomorrow I'm at SFU (the university across town), talking to their evolution group about why I'm working in a physics lab, and picking the brain of the Physics Dept's colloquium speaker.
Real tough guys - lichens
3 hours ago in The Phytophactor