Week 4: More qPCR and some Fluorospectrometer Frustration

When I analyzed last week's qPCR data, I found that the efficiency was only 85%, which was below the value we were hoping for. So this week I got to practice some more qPCR, and hopefully when I analyze the most recent data, my efficiency values fall in a more acceptable range.  We also started planning for RNA extractions next week, which involve a lot of solutions that need to be prepared, so I took the time to plan out all of the solutions and calculate all of the necessary volumes and such.  Furthermore, I was given the job of trying to figure out the Nanodrop Fluorospectrometer, which proved to be much harder than I initially thought.  In essence, one has to make standards and sample solutions containing a fluorescent dye; in this case we used PicoGreen, which binds specifically to dsDNA.  After setting up a standard curve, one places the sample on the pedestal, and the machine shoots LED light through the sample; upon excitation, any emitted light from the sample is captured by the spectrophotometer and quantitative data is collected.  We decided to test some standards of known concentrations to see if we could get the machine to deliver accurate values because if we could get it all working right, the fluorospectrometer would prove a way to obtain more accurate measurements.  We didn't have a written protocol for use of this machine, so I first had to skim through the user's manual, and then find directions on how to get readings with our particular Quant-iT PicoGreen dsDNA Assay Kit.  I found some directions online, and then commenced to adjust the volumes and plan out preparation of the standards and such as needed.  After creating the standards and sample solutions and running them through the machine though, the readings did not seem to make much sense because three of the samples had the same readings, which seemed to indicate some sort of error.  Therefore, the next day I attempted it one more time, and after some struggles with the machine and having to remake the standards, I finally got a nice standard curve and some promising results.  I have yet to analyze the data though to see if this frustrating process yielded accurate values.