Your Promega Connections bloggers were sitting around reminiscing the other day, “Back when I was in the lab…”. Kind of like Thanksgiving Dinner among your elderly relatives, it wasn’t long before we were one-upping each other with horror stories from our days at the bench–stories that included escape artist rats, a leaky sequencing gel apparatus, and the iconic radioactively contaminated post doc.
We decided to turn that conversation, with a lot of help from our favorite science cartoonist Ed Himelblau, into retro Halloween costumes based on our memories of things we used to do in the lab that don’t seem like such a great idea now. Enjoy…and if you have a few retro horror science costume ideas of your own you would like to add, feel free to comment.
Before the days of Wizard®, ReliaPrep®, Maxwell® and other systems, there was cesium chloride. You want super clean DNA for an electroporation or transfection? Purify it over a cesium chloride gradient. Spin it in a swinging bucket rotor in the Sorvall®, then carefully place your tube with its suspended bands of ethidium bromide-stained nucleic acids in a tube holder supported by a ring stand suspended above a UV box all in a dark room. While holding the back of the plastic tube with one gloved hand, shove a hypodermic needle through the front—hard enough to puncture the plastic, but not so hard that you stab your hand holding the back of the tube and inject yourself with ethidium bromide-stained DNA from the evil pathogen of your choice while your UV googles fall off and you blind yourself…
Second: Radioactivity. All of it.
Before fluorescent probes, His tags, and luminescent reporters there was radioactivity. Northern blot? Here, put the blot and your probe in an open Pyrex dish on an orbital shaker. Radioimmunoassays? You’ll need to have your thyroid scanned periodically. Gearing up could be a major undertaking: Double layer of gloves, lab coat, eye protection, dosimeter and for radioactive nucleotides emitting beta particles, a nice thick Plexiglass shield behind which to work.
Granted Sanger dideoxy sequencing was an improvement of the potential explosions that could accompany Maxim and Gilbert, but it still had its issues. For starters, there was the whole “unpolymerized acrylamide as a neurotoxin” issue.
To get maximum resolution, you needed long reads, which meant long glass plates—with really thin spacers that could be easily bent and ruined. Your glass plates that had to be squeaky clean—so that the acrylamide poured smoothly between them without bubbles and the gel would separate easily when the plates were cracked open at the end of the run. These were the days of bubble getters. And, in budget conscious labs, you reused the plates as much as possible, strategically combining chipped plates so that the chips were only on the outside on opposite corners. Of course the plates were usually siliconized, just to make handling them even more fun. And for people who worked with gel scanners, cleaning was ever more delightful, because you had to clean away spots that only your scanner could see.
Finally there was the sequencing apparatus itself. Last one in the sequencing lab gets the leaky gel box–and a timer going off every hour or so reminding you to refill the buffer chamber.
Fourth: Phenol:chloroform extractions.
Ah, there is nothing like the smell of phenol in the lab! (Unless it’s BME.) Using a mixture of the organic solvents yellow phenol and clear chloroform, you can take your restriction enzyme digestion or other completed reaction, add an equal volume of phenol:chloroform to remove the proteins and enzymes from your DNA or RNA. First you would mix the contents by vortexing then centrifuge to separate the mix into layers. You had to carefully remove the top, clear aqueous layer without disturbing the gunk (the protein and other debris known as the “line of death”) sitting on top of the organic layer. Once you were done, you had to dispose of the used phenol:chloroform. Usually that involved dumping the organic solvent into a larger capped container until full and someone carted it off to hazardous materials for disposal. Messy, stinky and challenging for those with shaky hands.
To ensure your insert was expressed correctly, it needed to be cloned into a plasmid vector in the sense orientation. That could be challenging enough when the insert had directional arms (e.g., BamHI and EcoRI digest on each end). On a blunt-ended DNA fragment, there is nothing to orient the insert so that the sense not the antisense orientation is achieved. And being blunt ended, the inserts and the vectors could ligate to each other, leaving the poor scientist with a mess of concatenated vectors or several inserts in a single vector to test for and sort out. But, achieve success with your blunt ended cloning scheme? You were molecular biology royalty.
Sixth: Indiscriminate use of ethidium bromide.
It seemed like ethidium bromide was everywhere in the molecular biology lab—from liters of gel staining buffer to agarose gels themselves to the tips of the gloves that people used to open doors and darkrooms. A DNA intercalating agent, the ubiquitous EtBr binds and stains DNA. Anything that intercalates into the DNA molecule also has the potential to cause breaks in DNA—and problems down the road.
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