I haven't updated people in a very long while as to what I have been doing in the KochLab. Technically I haven't been doing anything in there at all. From January to June I have been working with a collaborator in their lab... the Osley Lab. Koch sent me over there to learn basic level molecular biology and to begin the process of shotgun cloning.
You can follow all of my notes at OpenWetWare, but I will sum it up for you here. To make a long story short, I digested the ENTIRE yeast genome (Saccharomyces cerevisiae) with both XhoI and EcoRI. I then ligated the resulting fragments to an unzipping construct designed by Koch. Now we are ready to unzip. Of course there is still a little bit to work out, and even more to follow up on, but theoretically I have unzippable DNA fragments. We won't find out till we try.
I have shared a presentation I made for the first ever KochLab Symposium (posted on SlideShare), basically because I am too lazy to upload any of the pictures I have made and this talk contains everything.
As I mentioned you start with genomic yeast DNA. I digested the DNA with the restriction enzymes mentioned. I also digested pBluescript with the same RE's. I ligated the resulting fragments into the plasmid. From here I was able to make clones of whatever fragments managed to find their way into the plasmid, using E. coli. Using the color selection of pBluescript, I selected about 20 clones which would be used for sequencing and unzipping preparation.
After extra the cloned plasmid DNA I digested each sample with SapI and NotI. From there I picked about 5 samples that looked different from each other. Even though there is a low chance that the same sequence of DNA would be 2 separate clones, it was best not to take the chance. After selection and digestion, we sent a small portion of the samples off for sequencing.
The remaining sample was ligated to an unzipping construct. The SapI digested fragments were ligated to a specially designed oligo with a SapI overhang. The NotI fragments, likewise, were ligated to an oligo with a NotI overhang. There were other differences, but they don't need to be discussed here. The oligos are special because they contain a digitylated site, a nick, and a biotinylated site respectively. This means that I can attach the DNA to glass using anti-dig, attach a microsphere (streptavidin coated) to the biotin, and unzip utilizing the nick between the two. Sweet isn't it?
Once the tweezers are completed and calibrated we will test my work to make sure everything is peachy. We are almost there and I'll update at the end of the summer to report our progress until that point. Hopefully by then I should officially be a Ph.D. candidate!