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I spent a large portion of a term at our collaborator’s lab learning DNA cloning tricks, and ultimately ended up creating a new plasmid called pPIU1 which I then transformed into E. coli. For long-term storage the standard thing to do is stick a bunch of cells in a glycerol solution and freeze them at -80 C. At such a low temperature the chemistry in the cells is basically stopped—they’re in a kind of stasis until warmed up again. They keep more or less indefinitely, and barring the freezer suffering a power failure and warming up, the frozen stocks are your dependable backup.
Except when they’re not.
When my own group’s wet lab got up and running, I brought over some of the frozen stocks of my transformed E. coli in a styrofoam box filled with dry ice (so that nothing would thaw during the trip across town). I used these some of these cells to make a large supply of DNA and left a couple of other tubes as frozen stocks. This was a few months ago, and this past week I went about growing up some of the cells again. Streaking out cells on an agar plate with the antibiotic ampicillin (pPIU1 has an ampicillin resistance gene, so any cell with the plasmid should be able to survive just fine) and leaving them overnight in an incubator at 37 C normally means you’re greeted in the morning by hundreds of little while colonies of cells. Instead, there were two. This wasn’t very promising, but if even one of the two colonies contained my plasmid inside, all would be well.
Only one of the two colonies was able to grow in a liquid culture left shaking overnight (also a bad sign), but at this point I may as well have kept going. I miniprepped the bacteria that did manage to grow for plasmid DNA, and it turns out they do have something inside of them, but it isn’t pPIU1. This mystery DNA must (well.. who knows at this point) contain the ampicillin resistance gene too, but where it came from and how it got in these cells I’ll never know.
At this point I started getting worried that all my hard work from a half year ago was about to go to waste, but I luckily still had a lot of the plasmid left over from the supply I made when first bringing the cells to our lab. All I needed to do is get some E. coli from one of the other labs in the molecular biology department (thanks Paetzel lab!), and transform them with my plasmid again—something that should really only take an afternoon. Unless, of course, it doesn’t work, and for no obvious reason. This story is starting to go from comedy to tragedy.
I had checked to make sure the plasmids were still alright by running them on a gel after cutting them up with endonucleases. The bands were the right lengths, so I took that to mean the DNA was fine. But when I carried out the heat shock transformations (where you briefly shock the cells with high temperatures to make their membranes permeable to DNA), nothing grew.
For safekeeping (and what was now my last line of defense) I had left a number of tubes of frozen cells back at our collaborators lab. I called them on Friday and asked if they’d plate a few of the cells to see if anything would grow there. I headed over to their lab yesterday afternoon and was delighted to find that they seemed to grow just fine! While I can’t explain why I was unable to transform new cells with the same plasmid, I’m not going to worry about it. It may not be very scientific of me, but at this point I’m just glad that something has worked.
Biocurious is written by Andre Brown and Philip Johnson, since 2005. Content of the weblog is licensed under a Creative Commons Attribution-Share Alike 3.0 License.
