I witnessed this ground breaking, the culmination of SO many hopes and dreams for so long. Oxford college not only received a new library but got their dream of a new science building debt free. Brenham, one of the oldest dorms buildings, will be torn down and replaced with this new masterpiece. It will be the largest building on campus, with research laboratories and plenty of room to house the next generation of eager, young scientists. I even got to shovel a load of dirt myself! THAT I must say, was a real thrill.
*for the non-technical version and description of my project, see here.
I discovered a flaw in my design!
Some things changed since I last wrote! My goal is still to perform an in vitro splice reaction with rtcB (a ligase) and DNAzymes, to prepare for doing it in a cell. I reached a snag - a critical oversight. The substrate I designed was flawed. After the DNAzymes digested it, two of the pieces involved were the same size! Thus, the splice products would be indistinguishable. I was SO horrified, let me tell you.
Resolution: I may have dodged a bullet
Even though this was a terrible setback, and at first, I was really, really upset, I discovered two things that made it less painful.
THING 1: Integrated DNA Technologies (IDT), from whom I buy my DNA, has a special order one can place for long substrates that I didn't notice before. It's cheaper!
EXPLANATION: Long substrates are expensive to purchase because synthesizing them is hard. And mine is long. The first order I made for the substrate I'm using was over $100, because one can only synthesize about 200 nucleotides before synthesis is prohibitory. If in each addition of a nucleotide one has an 80% reaction yield ... well, reaction yield quickly goes down exponentially the more nucleotides one adds. The yield has to be nearly 99.9% to get anywhere. IDT somehow has another option called "ultra" that lets me buy a tiny amount of substrate for much less - $81! I was overjoyed.
THING 2: Another good thing that fell out was that I decided to order DNAzymes from the literature and build my new substrate AROUND them, instead of designing new ones myself.
EXPLANATION: This was for the reason that, though one might use the same DNAzyme reactive core, different binding arms can affect the reactivity. I didn't know that two weeks ago. Researchers have done some kinetics studies on some DNAzymes already and I can know these will be more reliable. I picked some of the fastest ones I could find that would also cleave the most RNA. It wasn't easy to pick, but I did my best.
The course now - onward charge!
My new substrate pieces now will be easy to tell apart and I have some new DNAzymes that will potentially work better and faster! They came in last Friday. I also ordered the parts of my substrate separately and made a ladder of them, to compare on a gel. Next week will be a REALLY interesting week!
What I was up to this past week
Week (May 5th – May 9th)
M-Tu: Literature searching for new working DNAzymes and designing the new substrate around them. Ordered the new DNAzymes, substrate and splint on Tu.
W: Ordered DNAzymes with eight nucleotide arms. Literature searched and planned for future experiments. Helped Weiwei run a gel. Started on completing Quartzy inventory.
Th: Ran an rtcB protein gel with the cell pellet and lysates. The protein had precipitated though. My substrates came in. Worked on the inventory.
F: Nanodropped all my stocks. Built my DNA ladder. Made fresh stocks for use and Nanodropped those. Imaged my protein gel and dried it on a frame over the weekend.
What's going on in my corner of the science world? Find out here!
Rolling Statuses: Technical journal blog. Here you may discover what the daily life of a grad student looks like: day-to-day snippets of life, clutter, rolling statuses and unimportant fluff.
Progress Updates: Will include entries with more meaningful science.
Weekly lab report: My write-ups on what I did each week (I posted these publicly during my rotation but not as much now. That may change.)
Here is a link to collected writing, poster and presentation tips.
As of February 8, 2014 I have officially joined the Salaita lab!! Very exciting. Stay tuned for updates. "Micro Min" category equates to grad school journaling; most of these have moved to my status updates blog under Home tab. See "progress updates" on this blog for more important news.