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A real pain in the USS

by PhilipJ on 5 June 2006

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.

  1. Uncle Al    4428 days ago    #

    Dogma, Metatron (Alan Rickman), “If you were to hear the real voice of God your soul would implode, We went through five Adams before we figured that out.”

    Did you talk to your cells?

  2. PhilipJ    4428 days ago    #

    Begged, pleaded. Offered them lives of luxury on beds of antibiotic-free agar, the loveliest of all 37 degree incubators, near the door with a view. No dice.

  3. several possibilities    4423 days ago    #

    Did you run a control with a plasmid you know will work? Several possibilities: Your competent cells aren’t really competent (could have closed up if chemically comptent or outright died if electrocompetent), you heatshocked for too long, you left the cells out in the warm for too long before you tried to transform. How long did you let them recover? And were you using plain-old LB or something rich like SOC media?

    One time I accidentally plated a vial of X-gal instead of my recovered cells. I wondered why the stuff I plated had a strange consistency on the plate. Then I wondered why nothing grew (and why I had these wierd crystals on my plate). Then the next day I saw the empty x-gal vial in the waste and the bacterial vial on the bench.

    Debugging is a pretty key skill. It’s very tempting to move on, but catching problems and being observant will help you in the long run. Experiential knowledge will tell you when some new observation is important, even in some of the most prosaic of techniques.


  4. philipj    4423 days ago    #

    Hi Isaac,

    I did the transformations both with my plasmid of interest and with another lying around the lab which we know also has the amp resistance gene. It transformed fine, hundreds of beautiful little colonies, while the pPIU1 plasmid didn’t end up getting taken up by any of the cells (or at least, I saw no colonies). So my cells were definitely competent, and why they wouldn’t take my plasmid is still a mystery to me. I was using plain old LB as my recovery medium, and left them to express the plasmid for an hour and a half before plating. My heat shocking was for a minute on the nose, which I think is a fairly safe period of time.

    I’m actually alright at debugging these things, but I’m really at wits end as to why the transformations with this plasmid didn’t work. The microbiologist we’re working with told me (after I had gotten frozen stock from her lab that were growing just fine) that one of the most valuable lessons she learned in grad school was not spending time on mysteries which ultimately aren’t that important to the end goal of the project, and given that cells from her lab were okay, that I should maybe keep going. I’d definitely like to know why this plasmid in particular wasn’t taken up, but I think it’s also a better use of my time to try and keep moving forward.

  5. several possibilities    4423 days ago    #

    One of the most important times in my grad career was when David Collum came to give a “fire and brimstone” talk about how one should do chemical synthesis. He described how in his laboratory he owned a -80 freezer which was dedicated to recrystallizing alkyllithium compounds. A grad student asked him why he did this (it was overkill), and his answer was that he did this to make the protocols hard enough “to keep the riffraff out”. The point was that science, and particularly modern, interdisciplinary science is full of dilettantery. And the only way to avoid being a dilettante is to be grounded in all of your techniques; with a comprehensive understanding of why something should work.

    I’m not quite done with graduate school, but looking back on things, the most important thing to me so far has been figuring out why things haven’t worked. Ideally, grad school is a training ground, where you are supposed to screw up. Maybe in a few years my opinions will change.

    All I can say is that several times, postdocs have told me to give up on something, and being bull-headed and chasing it down has led me to find some new things. Now I also know a handful of grad students who got lucky by publishing a paper or two and latching on to other influential papers—without developing their technique or garnering a sense of true, critical thought. These grad students irritate me (since they devalue my degree) and I can only prognosticate failure in their future.

    Who knows, perhaps they will be successful like James LaClair.

  6. several possibilities    4423 days ago    #

    For example, just the other day a I consulted a postdoc on an unusual product emanating from my LC/MS data; he suggested I throw it out since it had the wrong mass. Careful examination indicated that it was a adduct that could be easily recycled and considering my material runs about $15k/gram, I probably saved myself about $200 by not chucking it, and learned something in the meantime.

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