Fluorometer angst

As I predicted, I haven't gotten as much done this week as I would have liked but overall I have made progress. I successfully taught Cho how to electroporate bacteria and plate them on Amp-LB plates. He did a good job. Today I will teach him how to inoculate growth media so that tomorrow we can isolate his DNA.

A couple of months ago I bought an instument called a fluorometer. It uses fluorescent tags to measure protein, RNA, and DNA. Most fluorometers are very expensive ($10,000-$20,000) but this one is a simple unit that measures one tube at a time. It seemed to meet our needs perfectly so I spent the $700 our sales rep asked for. Sure enough, the Qubit (that's the model name) is easy to use but I have been having second thoughts about its accuracy. After measuring protein levels, I adjust each individual sample so that I am loading the same amount of protein in each lane on my gel. However, when I go back to the gel and stain it with silver or with coomassie blue, I am finding that there are different protein levels. As a notoriously poor mathmatician, I went back over my number to make sure I calculated everthing correctly - I did. This means that my raw data is incorrect. I wonder if there is anything in the cell lysates that autofluoresces? You would think not. If so, that is a problem that should have been addressed long ago by the many instrument manufacturers. I ran a protein assay again yesterday and am going to run four gels today. The first two I will stain with coomassie blue to double check my protein loading. If my results mirror my calculated data, I will rerun the gels this afternoon and prepare them for Western blotting. If they don't agree with my calculated data, I will have to make the necessary adjustments by visually gauging how much to load. Then I can rerun them. Enough writing. I have a lot to do.