Cell and molecular biology is a bit of a dark art. The way we perform our experiments has been passed down from generation to generation in sacred texts known as “protocols”. Like any recipe used and abused by generations of creative people, the methods and materials are continually refined as people try slightly different conditions, either in the hopes of improving the result or (let’s be honest) finding a shortcut allowing them to escape to their local pub before closing time.
Protocols used to be on paper (stained, torn, smeared, possibly slightly radioactive) – photocopies of photocopies with layers of marginalia successively enshrined – but are increasingly electronic. Either way, they represent an astonishing living record of scientific processes in action. They also unwittingly betray a little bit about the researcher who first composed them. One of my favorite bits from a protocol about isolating genomic DNA from Drosophila, written originally by a wonderful Chinese PhD student in our lab, starts off like this:
1. Put flies in tube.
2. Kill with stick.
I was thinking about protocols the other day when trying out a new method for culturing cells derived from the human retina. Cultured retinal cells, not surprisingly, don’t have all the characteristics of their tissue of origin, even when you genetically engineer them with some key genes. But someone recently discovered, and published, that if you add a smidgeon of sodium pyruvate in a slightly different formulation of standard culture media, the cells start to transform just a little bit closer to what they should be. Sure enough, a few days after adding the humble salt, my cells began to take on a telltale graininess: dark pigments, expressed by cells fooled into thinking they should prepare to absorb light.
“Look, they’re making eye contact,” quipped one of my colleagues as he peered down the microscope.
I am often astonished by the weirdly arbitrary nature of the procedures that have stood the test of time, and wonder how they came about. For example, when you want to zap DNA into a bacterium to make more copies, you perform a simple but very precise protocol that includes a heat shock of exactly 42 degrees Celsius for exactly 45 seconds. Who was the first person to ever work that out, and how many iterations did this molecular biology pioneer attempt before stumbling onto the perfect formula? And is it too late for me to buy him or her a drink?
I don’t know about you, but sometimes it all seems rather eye-of-newt and tongue-of-bat.
You know, its actually worse than that. It’s 42C for 45 seconds using the correct kind of tubes, the ones we used to call “15 mL Falcons” because that was the brand. And if you use a cheaper brand of tubes it doesn’t work so well. The reason is that its not just about getting the cells to the right temperature but also that the heating and cooling profile is right.
One of my students once inadvertently did the experiment. They hadn’t realised that the tubes were important and spent a long time trying to optimise the transformation because we needed lots of colonies (directly evolution experiment so each colony was notionally different). They’d been using eppendorfs (or rather cheap brand microtubes). We shifted to proper Falcon tubes and the efficiency went up 20-fold. Transformation really is a dark art.
Wow – I always use Eppendorfs! Your tip has just been scribbled onto my memory (since transformations are one of those protocols I now do from memory).
exactly 42 degrees Celsius for exactly 45 seconds
I’m sure there must be an equation, probably involving Planck’s constant and Avogadro’s number, that explains it.
If you’re using eppendorfs then it would probably not make much difference if you just do your heat shock at 37C and then dump in the media. To be fair most competent cell stocks these days are efficient enough that as long as your ligation/insertion/whatever fancy incorporation techniques young folks use today is halfway decent you’d still get a reasonable number of transformants. The home made stocks we made up were a bit of a different story. 🙂
I always had too many transformants, so 37 for 5 minutes in Eppendorfs was fine. Plus or minus a couple of minutes.
I like the thought of killing fruitflies with a stick. Priceless.
Actually Cameron, we used to make our own stocks. The trick was the freezing–liquid nitrogen rather than dry ice/ethanol gave a 5-10x increase in efficiency.
It’s all voodoo, but runs off the principle that “if it ain’t broke, don’t fix it”. All my non-scientist friends are amazed when I tell them quite how hidebound and even superstitious we can be about sticking to our (occasionally bastardised) protocols.
Out of interest it is amazing how you can see divergent evoulution in some protocols. My EM colleague here was taught EM by, let’s call him Prof. Y (I was going to call him Prof. X but then you would imagine him in a wheelchair reading your thoughts and telling the other X-Men what to do). So, she learned as one of Prof. Y’s later students, and I learned from Dr. Z who was one of Prof Ys earlier students. Our protocols are broadly similar, but there are distinct differences in how we embed samples, and how we heavy metal stain and wash them. Already I have made my own tweaks to protocols too.
How many scientific generations do you think it would have to be before there is a true schism in protocols in some sort of quasi religious fashion 😉 ?
How many scientific generations do you think it would have to be before there is a true schism in protocols in some sort of quasi religious fashion?
I don’t know, but they’ll be armed with Gilson p1000 pipettemen – one side with blue tips purchased from Starlab and the other, from ThermoFisher.
To broaden this discussion a bit, I remember being shown around some ICI Paints factory (back in those golden days when ICI was a proud UK company), and discussing paint formulations. It may not have been eye of newt but when I asked questions like ‘why do you have hydroxypropylcellulose in there?’, the answers were essentially it was added in to some previous formulation and no one had ever taken it out again although there was no obvious reason for its presence. So, formulations got ever more complex because nothing that had once been added to cure some long dead issue was ever subsequently removed, only new chemicals added. I suspect this is much commoner than one might think.
Thanks for this post Jenny. It explains a great deal. I was put off forever from this kind of lab work by precisely this worry – these formulations seem so precise, and yet so arbitrary, that I would never, ever be able to design my own experiments, ever, or so I thought. I honestly didn’t know, until reading your post, that protocols are handed down rather like Mosaic tablets or books of spells, rather than created rationally. Looking at bones seemed so much more straightforward. So I became a palaeontologist.
Very nice post, Jenny.
In my old lab in India, one test to measure if you are up to the mark was to have any new person make ultra-competent bugs. If you could make chemically competent bugs (Inoue method) with superhigh transformation efficiency (10 to the power of 8), nobody questioned anything you did after that. These bugs when snap-frozen in LN2 were the very best and we had plans to start a company to sell these bugs!
And yes, I had come across “use 15 ml Falcon tubes for heat-shocking” about 11 years ago and had realized the difference it makes if you switch to eppendorfs.
Makes me happy to be in electrophysiology where it’s possible to be a bit more rational (mostly)
Pretty sure it’s based on chemical principles coupled with lots of trial and error. Stem cell differentiation protocols are the same; based on signaling principles, coupled with years of trying different combination. And still, you need to make sure the cells sit still for 30 sec and don’t swirl the plate or it’s all gone Pete Tong. Lots of little details….
Jenny – you sure that’s not a copy of Advanced Potion-Making you nicked from a certain Mr. Potter? Those marginalia look creepily familiar…
Athene – you are *so* right. I once asked a good friend, and at the time senior (compared to me) graduate student why there was so much salt in Southern blot neutralizing solution. His reply was “it keeps everyone happy”.
Molecular biology is rife with this kind of thing. Particularly bad (along with transformation as discussed above) are PCR (should I include gelatin? DMSO? BSA? PEG? powdered lacewing flies?) and everyone’s favourite bugbear, ligations. People I knew used to swear that DNA ligase works best at 12 Celsius. Others claimed that 12 C was “the” temperature simply because it’s (roughly) the temperature of the butter-keeper in the door of a standard fridge, and someone in the mysterious past had gotten one to work by sticking it there overnight. Still others claimed that DNA ligase will have completed its job in the first five minutes or so, so leaving ligations overnight was a silly waste of time (I can confirm this to be absolutely true, at least in my hands, fifteen years ago, in a different lab than the rest of you, sometimes).
Another ligation gem was developed by a postdoc I worked with, who reckoned ligase has an optimal temperature… so he would start his reaction in a beaker of water at room temperature, which he’d put in the fridge overnight. His reasoning (if you can call it that) was that somewhere along the way, as it cooled down, it would pass through the optimal temperature and voilà! the ligation would work.
Forgot to say: many lab protocols would be improved with by the inclusion of that phrase, “kill with stick”. Just sayin’.
I used to set up my ligations on ice, leave them for an hour at RT and then stick them on ice again for that very reason.
Also, didn’t someone in this very salon once mention someone losing a ligation tube for a particularly recalcitrant ligation, then finding it a week later, transforming it and finding that it worked?
Great post! I inherited a protocol to lyse yeast by grinding them into a powder that declares the process complete when the resulting powder takes on a fine flour/talcum consistency. Now I pass on this protocol by asking the grinder to imagine sticking their fingers in white bread flour and assess yeast lysis by feel. Incredibly, this really does work well.
This was just to say that it isn’t molecular bio that has hard to assess protocols involving a bit of voodoo.
I have it – either reagents are designed to be used under an unbelievably wide range of conditions and/or not necessarily the conditions advertised. I’d like to call, as my first witness, an Arab donkey.
Many years ago when the world was young (OK, so it was 1985) I spent a couple of months in Israel as a tourist and
kibitzkibbutz volunteer. One day in Jerusalem, indeed it was on the Mount of Olives, Yea, even unto the Garden of Gethsemane, I found that I’d run out of film for my wee Kodak Instamatic.Just at that moment a donkey hove providentially into view, guided by an Arab merchant of film for said Kodak Instamatic, which he sold me for a fistful of $$$. Now, that film had been cooking in the panniers of that donkey for who-knows-how-long, such that by the time I got it, it had exceeded its use-by date by a more than a year. I kept snapping nonetheless, and guess what? The photos I got from that film had the richest, most vibrant colours I have ever seen.
I always secretly suspected that whoever decided to put herring sperm DNA into hybridization buffer was a bit of a perve. Or at least, the person who decided that herrings were worth milking that way. 😉
@Jenny – sheared human placental DNA. I rest my case.
I have always been slightly disturbed by the fact that anybody would have performed the “frog test” for the first time.
http://www.pjonline.com/blog_entry/glow-worm_frogs_toads_pregnancy
It’s also interesting that different disciplines have taken their own sweet time catching up with biochemistry. For example, as a student years ago in an immunology department I began to question why “lysis buffer” to extract proteins from mammalian cells was made with 300 mM salt, rather than the more isotonic 150 mM. No good answer was ever given to me. After months of thinking and searching (“research”) I figured out that initially the cells were being”lysed” as a pellet with a specific volume, and that the 300 mM NaCl was being added at a ratio of 1:1 to give a final concentration of 150 mM salt. That made sense. But in the meantime, the cell pellets were being spun down with no remaining volume leaving a final concentration of 300 mM. And that’s how the technique was taught to me.
It might please you to know that I had quite a nice conversation relating to this blogpost on twitter last night. See here: http://storify.com/bronwenann/conversation-on-protocols-in-the-lab-and-the-kitch
@ricardipus – a touchdown ligation! Oh I’ve heard everything now, lol.
There is a good dose of rhetoric and stoicism with some protocols though, look at how fervently people stick to Tris-based electrophoresis despite improvements.
There is also Schenk & Laddaga’s protocol for the electroporation of DNA into S. aureus (Schenk & Laddaga, 1992), which is so comically absurd it’s a wonder that hopping on one leg with a rabbit’s foot tied to your forehead isn’t part of the protocol. Needless to say it’s merely the yeast extract concentration that’s important.
I had massive trouble in a postdoc working on S. aureus electroporating DNA in, and quite frankly I’d have hopped on one foot with the whole rabbit tied to my forehead to get it to work!
Having said that it is I that was guilty of the “lose your ligation tube for a week” protocol for successful ligations.
ah, sorry I couldn’t remember who it was, Ian.
I was once put in charge of training a new grad student who was intent on improving all the lab’s standard protocols – an obsession he may have acquired while trying to beat the lab’s record for taking 24 samples from bacterial culture to minipreps to restriction enzyme digests to a photo of a nice clean gel (we beat the monotony of miniprep days by racing against the clock and updating the current record on the whiteboard if we beat it). While this zeal for improvement was admirable, and a welcome change from the standard practice of following the ol’ faithful protocols to the letter, I did have to work to persuade him that he should at least learn the standard protocols before trying to improve them…