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\nCloning panache<\/strong> Sometimes it’s cool to be old-skool<\/em><\/p>\n
\nThis week, it’s become clear that I’m moving into a distinct cloning phase. Of DNA, as opposed to of animals (or of an army of evil minions \u2013 although they might prove useful for analyzing my 20,000 movies<\/a>.) <\/p>\n \nNow, this is pretty familiar territory \u2013 I spent almost my entire PhD stint cloning constructs. Things were pretty primitive back in the early Nineties. There was only one sort of Taq polymerase, and you purified DNA using phenol, or on cesium chloride cushions if you were really fussy. You made your own competent cells. You extracted fragments from gels using low-melting agarose. You sequenced your final product by hand \u2013 with radioactive 35-S. If you had something really huge to clone, like a retroviral genome, you had to clone it using lambda phage \u2013 oy vey<\/em>, was that a pain. If it was small, you used M13 phage. (For my younger readers, imagine a tape cassette \u2013 this sort of palm-sized plastic thingie that could store about an hour of music. Cassette tapes are to MP3 players as M13 or lamda cloning is to the current methodology.)<\/p>\n \nIn the meantime, some things have changed. And not all progress is unwelcome. Yesterday I made a construct using the Gateway system \u2013 I don’t like to give excessive airtime to product placement, but this nifty system does away with the classic cut-and-paste ligation in favor of a snazzy recombination job on vanishingly small amounts of starting material. (This means you can usually clone directly from your collaborator’s miserly gift of 0.0001 microliters of DNA without having to amplify first: bonus.)<\/p>\n \n“How many colonies should I pick for the minipreps?” I asked one of the youngsters in the lab. “Six? Twelve? Twenty-four?”<\/p>\n \n“How many?” He blinked at me, uncomprehending. “Just one. They’ve all got the insert, all in the right orientation. Otherwise they wouldn’t grow.”<\/p>\n \nNice. I can live with that.<\/p>\n \nBut other things, reassuringly, haven’t changed. In fact, my knowledge seems a sort of forgotten, retro-chic dark art that is coming in extremely handy amongst the younger lab denizens when their turbo-charged, kit-driven cloning doesn’t work the first time. Some of the tips I’ve passed on (to frank adoration) include using the Klenow fragment to help finish off the ends of PCR product, for when that swanky new brand of Taq gets too lazy to complete the restriction tail you’ve engineered in; ethidium bromide spot plates, handy for quantitating tiny amounts of insert and vector when the molar ratios really matter, as for three-way ligations; the concept of low-energy hand-held UV illumination that doesn’t damage your DNA fragments while you’re cutting them out. These are all things that I would have thought I’d long forgotten, but which seem to have become ingrained when I wasn’t looking. It is almost as if, after returning to the lab after my career break, I had to go through that distressing one-year period of almost complete lab amnesia before being able to retrieve all the stuff in long-term storage.<\/p>\n \nKids these days? They just don’t understand.<\/p>\n","protected":false},"excerpt":{"rendered":" Scientific methodology seems to come in distinct phases for me. One month I’m knee-deep in biochemistry; the next it’s all confocal microscopy on cells, or annotating images onscreen. This is part of what I love about research: the familiarity of … Continue reading