Analysis and Disappointment

As a quick reminder, my project is working to create a tool that can eventually be used to correlate transcription and translation within a cell (whether the number of mRNA transcripts correlate directly to the eventual concentration of protein output). I intended to use a reporter plasmid to track the concentration of protein, while thinking about other tools to tackle the other side of the problem – identifying the concentration/number of transcripts.

This reporter plasmid was donated to our lab from another lab, and had been isolated previously – I went back to a glycerol stock of the bacteria (bacteria containing the plasmid, combined in equal parts with glycerol, then stored at -20 to keep them stable) and started new cultures. My cultures grew to be very turbid – that is, contained a lot of bacteria – and I then conducted a miniprep. A miniprep is a procedure used to isolate plasmids from inside bacteria: the host bacteria was one that is normally empty of plasmids, and should only have had one plasmid within it, which was our plasmid of interest. The miniprep seemed to proceed very normally. I had a good concentration of plasmid, and it ran out on a gel as one very clear band: perhaps running a little high, but at least, cohesive.

I had also designed primers to amplify the plasmid once isolated. Here, the science began to unravel. PCR primers are typically extremely specific to the plasmid: they bind to only one location, and from there must yield only one, linear product. However, the gel image following PCR was a patchwork of one band at the expected weight, faint but visible, amongst at least four other bands at both higher and lower molecular weights. Unsettlingly, the strongest band was about twice the size of our expected plasmid.

However, that was still perhaps within the bounds of explanation. Perhaps the plasmid was forming some sort of dimer, with two copies of it clinging together in an odd hybrid at a heavier molecular weight. Well, a diagnostic digest should have set that straight. In a diagnostic digest, an enzyme which recognizes a specific nucleotide sequence is used to cut a piece of circular or linear DNA in one or more places. I used a combination of two enzymes (two cuts in a circular plasmid = two bands on an ideal gel) that should have yielded two easily distinguishable bands. Unfortunately, this was not the case. Regardless of whether I used both enzymes or just one, the plasmid only yielded a single band – at a very discrete molecular weight, which was in either case unexpected.

Essentially, the plasmid we had contained an expected sequence, but in multiple locations, and was about twice the weight of our desired plasmid…in short, not at all what we had wanted to isolate. What a let-down!