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 – buffed, polished and fully verified.
I quite like the thought of being a Cloner For Hire – blow into town with the tumbleweed, step into the bar and, over the sudden silence of the piano’s cessation, drawl slowly, “I hear someone here needs a GFP-tagged point mutant?”
As one, the hushed crowd turns to stare at the post-doc slumped over his whiskey in the corner.
It 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 – 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?)
A 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 – 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 “quite working” (a comment which would haunt me later). The strategies were pretty complicated – long-range PCR, intermediate subclones – hey, even a three-way ligation! But nothing I hadn’t seen before. I like a challenge. And it was heart-warming – he’d also stocked the yellow box with aliquots of everything I might possibly need on the adventure – ligases, buffers, nucleoside triphosphates, X-Gal, obscenely expensive turbo-polymerases. It was as pleasurable as receiving a picnic hamper from Fortnum and Mason.
Despite 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, “I will finish these clones before the broth runs out. Mark my words.”
When 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 – should I be worried?
Anyway, 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 – 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.
“This one is labelled ‘miniprep DNA’, but it’s a liquid at -20,” I told my benchmate, sticking the tube under her nose. “Does it smell like alcohol to you?”
“No,” she said slowly. “Actually, it smells like nectarines.”
“That was from my lunch, silly. How can we work out if it’s alcohol?”
“Color a bit with some dye and see if it floats on water?” she suggested.
“Good idea. Or – hey! Maybe we should try to set it on fire!”
And 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.
But the clock is ticking.
I’ve always felt a twinge of guilt when I’ve left a lab, hoping people would be able to pick up the project and continue. Although, to be honest, I don’t know how anyone can take over from anyone else without at least three month’s overlap, no matter how good your notes are.
The other thing is how quickly you forget. I was fielding requests even a few weeks after leaving my last lab, and it was already getting hazy.
How very timely a subject!
(Seriously, best of luck.)
Heather, I totally missed Anna’s post – thanks so much for pointing it out. Timely indeed! I love this: ‘never use a reagent you didn’t make or check in some painfully meticulous way.’ Even when they ask you to!
It was as pleasurable as receiving a picnic hamper from Fortnum and Mason.
Fantastic! That just made my day. How thoughtful of him! I hope you make good on your vow to finish before the broth runs out.
My PhD supervisor passed his l33t plasmid cloning skillz on to me, and it saved me so much time during my postdoc. I also got pulled in to help other people with their projects. No-one else in that lab was even considering the relative concentrations and sizes of the vector and insert(s) when setting up a complicated ligation, which was one of the most basic things I got taught!
Yes indeedy: x = v/(i)(n). I never ligate without calculating molar ratios. But you are right: almost nobody else bothers.
Molar ratios was the term I was searching for, in vain obviously. I’ve been out of the lab too long!
Are those fluorescent green thumbs that you have? In which case could I suggest using more gloves…(and removing your hands during the electroporation)?
I think, Stephen, that Jenny has been transformed with GFP. Biolistics into the hand.
Hmmm. Now that would be a cool tattoo.
It is sometimes possible to pass stuff along. I’ve heard back from the lab where I did my MSc, and apparently my thesis is the source of the procedures they’ve been using lately (they finally got funded to finish my project, three years later…) I was one of the only ones to write things done, and I’m one of those insanely detailed persons who puts every detail in, with any changes (working in a Big Pharma lab will indoctrinate you into signing every mistake with your initials!) When I left the lab, I also wrote up a table of contents for every lab book, documented every gel (with all the conditions to recreate them and every other cloning I did and then expressed to make my purified proteins. I had walked into too many other labs and projects where I was handed a few sheets of procedures and told to “go to it” that I was determined not to leave someone else in that position. I would like to think my karma will be doing well because of it…
Shall we say P20’s on Main Street at Noon?
I love, love, love cloning for other people – especially for German post-docs employed by cruel department heads who have been trying for months to make an expression construct that I whoop out in a week (with maps!), refuse glory, demand sushi…
And I never apply mathematics to my molar ratios – Like my cooking, I clone by instinct.
Ooh. A showdown between logic and emotion.
(I’ve tasted Audra’s cooking. To say more would be ungentlemanly).
Stephen, I’ve got an N-terminal GFP expressing in my left thumb, and a C-terminal one in my right. I adore the idea of a GFP/RFP tattoo (not on my own skin, mind) — it doesn’t seem so crazy. Give it ten years.
Kristi, I salute your transferability. I am also one of these obsessive notebook keepers, but it doesn’t come from my stint in industry. It comes from being a novelist. There is a lot of detailed pathos between my pages.
Audra, unfortunately I’m one of these annoying people who likes to follow a recipe. When I take short-cuts, I do it in a defined way, noted in my book, so I can repeat it if it works (and not if it doesn’t). The math comes in handy for trickier ligations — when you have hardly any insert, or when you are ligating more than two fragments. I pick a mid-point using math and then do a ligation with more and less insert as well in case my quantitation wasn’t optimal. I just find it’s more reliable than guesswork.
I adore the idea of a GFP/RFP tattoo (not on my own skin, mind) — it doesn’t seem so crazy. Give it ten years.
So. Fecking. Cool.
All I have to do is decide on a design.
Is there any molecule that fluoresces only under far-red? The clubs could start producing that wavelength in their lightshows. (Though I hate far-red tags. Bloody useless, really.)
Richard, I think you need a tattoo of your favorite kit. Obscenely strong magnet?
Jenny, anything fluorescing under far-red would emit out of the visible range, unfortunately. At least, I think that’s how it works!
I was actually thinking of something more subtle, and biological: stress fibres or something like that. Perhaps a dividing cell?
Clubbers could wear special goggles. It would be all the rage.
Which phase of mitosis? I sort of see you as a metaphase plate.
The prettiest phase.
Maybe something based on this?
Look at my ruffles
Um, that would be interphase. How long have you been out of the lab?
@Jenny – Is there any molecule that fluoresces only under far-red?
Your friendly neighborhood recent nobellist to the rescue
So with infrared’s penetration abilities, Richard could get that tattoo on his spleen. It’s amazing what you can learn from the literature!
Jenny, I know that’s interphase. There was a paragraph break, deliberately.
I’m a little concerned about getting my spleen tattooed. But perhaps there is a market for FP-laden soft drinks…? Oh, even cooler: i.v. injection! Your veins would glow!
Whatwever else you get out of this, Jenny, I think you have the start of a novel right there.
Dr Ginevra Rohne(*) works in a hot lab at the cutting edge of science. One day, a colleague phones in to say he’s got visa problems, and won’t be back for a couple of months … would Dr Rohne carry on his experiments until he can get back? Dr Rohne agrees, but the more she delves into the reagents and files left by her absent colleagues, the more curious the case becomes. Before she knows it, she’s trapped in a web of international intrigue, deception, scientific double-crossing and fluorescent plasmids. Only she can possibly find a way out – but she doesn’t know where to start. And the clocdk is ticking.
Surpasses Tolkien At His Best – Mallorn
More Erotic Than Gravity’s Rainbow – Dr R. P. G. of Rotherhithe
Excuse Me Madam, But Does This Bus Go To The Station? – Dr. R. O’H of Helsinki.
No resemblance intended, living or dead, blah blah blah
bq. Surpasses Tolkien At His Best – Mallorn
Heresy! Burn him! BURN HIM!!
Richard: Oi. And your point is?
Henry, you now owe me a new keyboard.
chuckle.
Mind you, the idea of a colleague stumbling on something in a departed researcher’s notebook is actually a great idea for a plot. I may have to ponder this more closely as I wait for my x-Gal/IPTG colonies to actually turn blue. They were pale white this morning, and only now are starting to take on a tinge of turquoise. Too soon to pick, though! It will be a race between the x-gal and the pub.
Mind you, the idea of a colleague stumbling on something in a departed researcher’s notebook is actually a great idea for a plot.
I shall expect to be acknowledged, mind.
Naturalmente. And I’ll see if I can work in a couple of chickens, just for you.
You’ll be careful not to over-egg this, won’t you?
Not ova-ly.
That pun was truly fowl.
Also – cloning for other people? Are you lot crazy?
*goes off mumbling about how if people can’t clone their own
shitthings, then they shouldn’t be blah blah blah blah mumble mumble rhubarb etc.@jennifer: “There is a lot of detailed pathos between my pages.” Well, there was a lot in mine, if you read between the lines. At least I wasn’t as bad as the other grad student in the lab, who insisted on keeping notes, and then writing in her lab book in LONGHAND. she would take days to catch up on a few months of lab experiments. She used to laugh that my handwriting is nigh unreadable (computers are my friend now), but my supervisor also got mad at her when I finished an exam and she still had two questions to go. He actually asked me to “translate my paper” afterwards but I still got the marks 🙂
@Richard – in my current job, you would be very, very scared to know the number of scientists I talk to daily who can’t even be bothered with a positive or negative control (and both in the same experiment are a rare occurence!) It scares me to know that the state of science in some labs…but they aren’t being published, for the most part (until I found a few papers that made me shudder).
… and people say we don’t need peer review…?
Kyrsten
Is there any other way to write a lab notebook but longhand?
I confuse my fellow labmates by using what Americans call ‘cursive’ (and what Brits call ‘joined writing’, although they aren’t really the same thing). As good as Leonardo’s mirror-image writing for code in this country!
The only controls I refuse to do are the ones that come in kits (e.g. TA cloning vectors, where they encourage you to blow £30 each ligation testing the uncut vector for ‘background’). I think it’s a scam to make us buy more stuff. But you are right – controls may be a lost art.
@jennifer: What I mean by long-hand is handwriting, as opposed to printing. Beautiful handwriting, but no place in a lab notebook IMHO. She was crazy with the whiteout too :O
I absolutely agree that most controls can be dispensed of. But I’m scared by those that refuse to do a negative control for a particular assay that requires you to compare enzymatic activity to a baseline, which the negative control provides.
I am also scared by those that change a particular procedure in 5 different ways, and then blame the assay because they’ve changed some major steps (and haven’t asked anyone if they are indispensable steps or not).
Peer-review is so crucial. While it doesn’t get rid of every problem (I’ve seen so many mis-citations of products such that if you bought what they “used” you’d not actually be able to replicate their work), it certainly pushes scientists to a higher standard.
I’ve probably said this before, but in the lab where I did a rotation at the beginning of grad school, the PI had put a sign over one of her students’ benches:
POSITIVE CONTROL
NEGATIVE CONTROL
EVERY EXPERIMENT
I’m also pretty sure that I once ran a PCR reaction with two test samples and eight, count ’em, eight separate control lanes. That was, however, about a million years ago. Controls were more important then.
Kyrsten: yes, I do my lab notebook in handwriting! I write faster in cursive than in print because the pen doesn’t have to leave the page. And it’s actually neater than my print — although, like many things, it’s getting a lot messier as I age. I think some people are confused by the way I was taught to handwrite the letter ‘z’.
I always do positive and negative controls in my actual experiments. Cloning — making tools — doesn’t count, I don’t think, as experimentation. They do become important when things aren’t working in that department, though.
Damn! I could not have read this entry at a more opportune moment! Thank you, Jennifer, for making me feel I am not all alone in the universe stuck between the Scylla and the Charybdis, with (a) an ex-labmember’s tome-size notebooks and (b) my PI’s instructions to make sense of his experiments in order to repeat one particular assay which seems to have worked out in his hands quite well and needed to work in mine. I finally did manage by using a judicious combination of skulduggery, legerdemain, psychic mind reading, a bit of voodoo, and as much native intelligence as I could muster (which wasn’t much). If only he had mentioned anywhere in his notebook that the assay required a special type of microplates…
I don’t cook for gentlemen.
Ligations – how nostalgic. I do all of my cloning without it since I discovered the joys of PCR mediated insertion. Ever so much more elegant.
Audra – do you have a recipe you could share? Oh yes, you don’t do recipes. Video diary of your next exploit?
(This is actually a serious question – I have no idea what you’re on about but would like to. Is it some sort of recombination within the PCR?)
One for JoVE?
Just had this crazy image of Audra as A-list JoVE celeb waving on the catwalk.
Think it’s time to access some carbon source.
Um. Quite.
Update: two of my clones safely to intermediate subclone stage (though still need to be confirmed by sequencing that there are no non-synonymous point mutations introduced by PCR. Anyone every used Pfx Platinum?). Third clone floundering due to continuing anomalous restriction of starting materials.
Is it Friday yet?
It’s Friday somewhere in the wor–
ah. No. Not quite.
Something along these lines perhaps, or these ?
I haven’t tried either, so can’t say for sure. But I use PCR and I do some cloning, and I play around with custom adapters to insert restriction sites that were not where I wanted them to be. I don’t quite see how to get around ligation.
On the other hand, I definitely use PCR to get around the necessity of cloning any sort of DNA template for riboprobe synthesis. Happy to share the protocol(s).
The Gateway plasmid system bypasses ligation in favor of recombination. But I don’t think it’s generalizable if you don’t have a vector with the correct recombination signals.
I am intrigued about that second one-stop-shop protocol, Heather – many thanks for the link.
I think some people are confused by the way I was taught to handwrite the letter ‘z’.
Hm. Try them out on capital “Q” then. That’ll make their heads spin around. At least it will if you do it the way I was taught (looks kind of like a curly number “2”).
Ah: there’s even “an animation”http://www.peterson-handwriting.com/animCrsvCap/Q%20crsvcapAn.html of what I’m on about.
Bugger. Forgot the colon.
Linky linky link
Hm. Tried to submit a correction, and it disappeared. Bloody punctuation.
Correct link is here.
You, eh, happy talking to yourself, Winty?
Mumble mumble mumble.
Honestly, the first correction failed to appear, then when I posted the second one, they both showed up.
I’m going back to bed now.
I write my upper-case Q’s like that too!
/warm fuzzy feeling
I went to an American school for a few years in elementary school, so I know the weird z, and the weird Q, but I have mostly shocked people in Holland with the capitals I and G, which are totally different. I unlearned them, but can still do them, and people don’t believe that Americans really write like that. =P
Hm. When you print, do you cross your z’s? That confuses people on this side of the Atlantic no end.
I cross my z’s in cursive and print.
Much less confusing that way. What about the number 7 when you write it?
I cross that too. I have very nice sevens. A lass at college once wondered what a country boy from Lincolnshire was doing with such cosmopolitan 7s, and then she discovered my background.
When you print, do you cross your z’s?
Only if it can be mistaken for a 2 (in abbreviations, gene names, postal codes, etc.)
I think I picked up the crossed 7’s and Z’s somewhere between accountancy and cricket.
After my stint at the EMBL, I find myself adding that extra stroke to the number 1 when I label things (like an upside-down carat). Another thing that I find really good is the comma for a decimal point – a little dot can be hard to see when scribbled on a tiny Eppendorf tube, meaning sometimes you’re not sure what order of magnitude a reagent is. But the comma is hard to miss.
I have very nice sevens. A lass at college once wondered what a country boy from Lincolnshire was doing with such cosmopolitan 7s, and then she discovered my background.
Interesting story… this lass, would she happen to be a certain “enthusiastic control freak, ready to organize your laboratory”?
@Eva – you probably put a stroke through your zero’s too, am I right? Or is it just computer science geeks who do that?
Same thing: only if it’s somewhere where it can be mistaken for a letter o. Like if I would write “w00t!” out in print (which I never do, mind you – I don’t even type it – but the only other example that comes to mind is a friend’s e-mail address, which I certainly won’t publish)
I always stroke my zeroes.
That just sounds dirty…
I am glad that someone is paying attention in the back row.
I used to cross my 7s, but stopped. Too European I feel. We had a Ukrainian postdoc for a while who used the European 1, that looks like a US 7 (sans cross bar). Confused the hell out of my mentor; took a lot of explaining because he simply refused to believe that there were different strokes for different folks
For things dated on or after the 12th of the month, the US vs European date order thing has probably spoiled a few Nobel-worthy projects in their day…