First piece of news this morning was from Cedric saying that the manufacturer had confirmed that they wouldnt come until wednesday, thus making us lose three days of experimentation which considering that i am only here for 9 weeks is a weighty sum.
Rachid decided we could do at least some rough tests using the UV lamp as our light source instead of the laser. We were also using a DNA that had been marked with a different fluorophore (cy3 instead of alexa) so we needed to change the filters that the light passes through so that only the wavelengths that it absorbs and emits at would pass through. Rachid spent 2 hours screwing and unscrewing different lenses , checking their wavelengths, and testing them with the new DNA. We couldn't see anything. The camera wasnt even working at first but after we rebooted and restarted the program, the DNA still didn't show up. It was clear that the light we saw passing through the lens was not as intense as it should have been but it didn't matter what combination of filters with appropriate ranges we tried, it was still impossible to focus on any plane.
Rachid even tried looking at it optically but had no success. So I have spent the afternoon reading some scientific papers instead. The first one was titled "Visualizing DNA Replication at the Single Molecule Level". It focused on describing the exact step by step instructions for two different methods used to observe DNA replicating itself. The first method bonded beads to the DNA that could then be directly observed with a CCD camera. Depending on how much the beads are calculated to move, the analysis can state how much the DNA has either lengthened or shortened. I am still unclear as to why the DNA would shrink or lengthen and what the correlation between either change would say about the way it replicated itself. The paper was more of a manual so it did not describe the theory behind this behavior or even what their conclusions were after performing the tests. The second method used TIRF! Instead of watching the beads, the growing length of the DNA is observed directly. In both cases flow chambers were used to prepare the surfaces with layers of Blocking Reagent and streptavidin. Their flow chambers consist of three layers, the glass slide, doublesided tape with a channel cut through it, and a slide made from Quartz. The advantage of this method of layer deposition seems to be better control of the uniformity of each layer, but I will have to ask Rachid to be certain.
In reading this paper I focused on where their slide preparation methods deviated from our own as well as where they were similar. Other than the use of flow chambers the other greatest discrepency was that they sonicated their slides to clean them while we use a plasma cleaner. In comparison to their protocol ours is much faster.
The second paper I read was entitled "Single molecule Enzymology" and was largely a discourse on the importance of single molecule studies. The authors put forward that most experiments can only observe the overall average result of an experiment are likely to ignore the small percentage of contradicting results. As a result of this, a lot of information is lost because although the property you are studying maybe something that ought to be uniform to the group it could also be a property of dynamic disorder ( one which fluctuates with time individually for each molecule). To illustrate their claim evidence from one experiment on the enzymatic turnovers of a single cholesterol oxidase molecule were described in depth.
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