Over the past few weeks, I’ve stepped in to help a colleague in need. I can’t go into any details, but let’s just say that owing to an astonishingly pedantic visa renewal technicality, a particular country’s immigration policy has necessitated the precipitous (but hopefully temporary) departure of one of our post-docs. He’s due back at the end of July to start a sabbatical position in another lab, on a very tight schedule. And he needs three new DNA plasmids to be ready on his return \u2013 buffed, polished and fully verified.<\/p>\n
\nI quite like the thought of being a Cloner For Hire \u2013 blow into town with the tumbleweed, step into the bar and, over the sudden silence of the piano’s cessation, drawl slowly, \u201cI hear someone here needs a GFP-tagged point mutant?\u201d<\/p>\n
\nAs one, the hushed crowd turns to stare at the post-doc slumped over his whiskey in the corner.<\/p>\n
\nIt made sense to ask me. This particular colleague has done a lot on my project and will be a co-author on the paper I’m finishing up now. And he’s offered to stick me on his paper, which is right neighborly. But more importantly, I’m the only one in the lab besides him who really does much molecular cloning \u2013 all the geneticists just muck around making their male flies do naughty things with virgins. But I’m old school, and it when it comes to subcloning, I tend to have a green thumb. (You can see where this is going already, can’t you?)<\/p>\n
\nA few hours before hitting Heathrow, the post-doc presented me with a beautiful yellow plastic freezer box containing everything I might possibly need to finish making his three constructs \u2013 he’d even crafted little machine-printed labels for the tubes. He was a third of the way through two constructs, though he did mumble something about not everything \u201cquite working\u201d (a comment which would haunt me later). The strategies were pretty complicated \u2013 long-range PCR, intermediate subclones \u2013 hey, even a three-way ligation! But nothing I hadn’t seen before. I like a challenge. And it was heart-warming \u2013 he’d also stocked the yellow box with aliquots of everything I might possibly need on the adventure \u2013 ligases, buffers, nucleoside triphosphates, X-Gal, obscenely expensive turbo-polymerases. It was as pleasurable as receiving a picnic hamper from Fortnum and Mason.<\/p>\n
\nDespite this promising start, it all started to go wrong surprisingly quickly. The tube that was supposed to contain half a milligram of gel-purified PCR product was, in fact, entirely devoid of DNA. The vector that was supposed to contain only one EcoRI restriction site appeared to actually have two. Another vector seemed to be contaminated with a smaller plasmid. Undaunted, I made half a liter of LB broth spiked with ampicillin and told my benchmate grimly, \u201cI will<\/em> finish these clones before the broth runs out. Mark my words.\u201d<\/p>\n \nWhen it became clear I’d have to start all over and venture into the post-doc’s personal boxes to find new stocks of plasmids and primers, and into his notebooks to cross-reference them, it was then that it truly hit me how difficult it is to carry on a project after someone else has gone. For in the privacy of one’s boxes, things can get very chaotic very quickly. We all do it: how many of us are guilty of filing away rows of minipreps or ligations that are only numbered, convinced we’ll remember what they are by where they are sitting in the box? It is so easy to cut corners on record-keeping when night is falling, when your timer is alerting you about your other experiment, when you’re late for lab meeting. It’s made me start thinking seriously about barcodes \u2013 should I be worried?<\/p>\n \nAnyway, the post-doc had pretty good handwriting, but he never put dates and it wasn’t always easy to tell exactly what sort of animal was lurking in the tubes \u2013 Fragments? Digests? PCR products? Miniprep DNA? I found at least three tubes that seemed to represent the vector I needed, but none of them looked quite right.<\/p>\n \n\u201cThis one is labelled ‘miniprep DNA’, but it’s a liquid at -20,\u201d I told my benchmate, sticking the tube under her nose. \u201cDoes it smell like alcohol to you?\u201d<\/p>\n \n\u201cNo,\u201d she said slowly. \u201cActually, it smells like nectarines.\u201d<\/p>\n \n\u201cThat was from my lunch, silly. How can we work out if it’s alcohol?\u201d<\/p>\n \n\u201cColor a bit with some dye and see if it floats on water?\u201d she suggested.<\/p>\n \n\u201cGood idea. Or \u2013 hey! Maybe we should try to set it on fire!\u201d<\/p>\n \nAnd so forth. The LB\/amp broth currently stands at 350 mL, and I’ve just retransformed every starting plasmid into bacteria from scratch, so at least I know what I’m working with. <\/p>\n \nBut the clock is ticking.<\/p>\n","protected":false},"excerpt":{"rendered":" Over the past few weeks, I’ve stepped in to help a colleague in need. I can’t go into any details, but let’s just say that owing to an astonishingly pedantic visa renewal technicality, a particular country’s immigration policy has necessitated … Continue reading