Jenny’s post last week set me thinking. I have, apparently, a reputation for being a bit down on kits. Can’t imagine how that got started. The truth, as always, is a little more complex than that. Certain people here might be surprised at, for example, my attitude vis-à-vis pre-cast gels.
First, some history.
Back in the pea-soupers of time, when I was but a sprog of a grad student, I learned the secret of what was in the mystical potions A, B, L, M and H. I made 50 ml preparations of these, along with 10 mg ml-1 BSA and 20 mM spermidine, properly sterile-filtered and everything: and this was such a huge amount that even when Boehringer started supplying buffers with their restriction enzymes I continued using my homemade stuff. Seemed to work, too.
I remember my first ever PCR. I spent a week preparing—under DNA-free conditions—all the buffers, from scratch. I remember being terrified I’d screw up and the experiment wouldn’t work (it did. Work, that is). And then there was the magic of 35S dideoxy sequencing, and the encroaching enkitment of that method, with colour-coded caps and a red dye in the hot stuff. Big sequencing gels, with degassed urea/acrylamide mixes and sealing the bottom of the plate with agarose. Frankly, despite the loss of this skillset, I certainly wouldn’t go back.
The restriction buffers, the magic 10 X PCR buffer (and even the pre-made MgCl2) stock; yay verily the Sequenase kits: all these supplanted homemade methods and reagents in their time. Overall, a good thing, I say.
And pre-cast protein gels? Anyone who has had to pour a slab of ten gradient gels, complete with stacking and resolving, and then clean up the semi-polymerized gunk all over the desk because one tiny little thing wasn’t nanometer perfect; or who has found bubbles right at the resolving interfaces making half of all the gels unusable— anyone, I say, would gladly spend a few extra quid on the pre-cast gels from Invitrogen. My boss in Cambridge actually instructed us to use pre-cast SDS gels because of the saving in time, reagents and neurotoxins. (Excepting native gels, but that’s another story and I won him over on that one, too.)
But there are—or would be if I went back to the lab—two kits that I baulk at.
The first is the Quikchange mutagenesis kit. I remember reading the paper in which the method was described, and comparing the contents of the kit with what we could put together by buying the reagents individually. I approached the boss with a proposal, we bought one kit and photocopied the instructions—the cycling times and temperatures and advice on choosing primers—and then bought Pfu (which always came with its buffer, natch) and dNTPs and Dpn I separately, at a small fraction of the kit price, for exactly the same reagents. Minus a cardboard box.
Me, not seeing the point.
The other kit I hate (I say ‘kit’, but really it’s the whole industry) is plasmid prep kits.
Take minipreps. Now, I’ve worked in the industry, and I know (a) exactly what goes into these kits and (b) precisely how crap they are. Back in 1999 Qiagen were selling minipreps at £1.09 per. The manufacturing cost was ~17p. That’s one hell of a markup. And they were shit. The standard protocol, if you followed the instructions, would take about 20 minutes. With the kit I made, you could do the same thing in 11. The Qiagen kit, if you followed the instructions, would give you nicked DNA and crapped out if you gave it too many bacterial cells. Mine wouldn’t.
Guess which wouldn’t sell? Betamax versus VHS, anyone? (Marketing, marketing, marketing. It’s so easy, except when it’s done by people who seemingly were lobotomized at birth.)
The thing, the really galling thing, is that you can get DNA good enough for screening colonies (and indeed, with a bit of tweaking, for sequencing) with homemade reagents at fraction of the cost, with the same (if not less) hands-on time, and without those tedious wash steps spewing gunk all over the microfuge that nobody, ever, cleaned up.
So I’m down on miniprep kits. I’m also down on midi- and maxiprep kits because, let’s face it, they are incredibly tedious because they still take half a day if not more and you still have to do a couple of alcohol precipitations and they are fucking expensive.
I say learn about the hazards of phenol, so that you get good and scared when you go into a lab (I made a med student cry once for not treating phenol with respect. But then, you’re talking to someone who before he started his DPhil knew a chap who spilled 500 ml of the stuff all over his legs and spent three months in Stoke Mandeville having skin grafts, and his kidneys pumped) so that when you have to use stuff that is really nasty you aren’t totally unprepared for it (because, for example, I don’t think there is any kit version of paraformaldehyde).
It’s not because you might get asked about the chemistry of these things in your defence. It’s because some things really are a waste of money and time. Other things—like Jenny’s bacmid kit, or those complicated expression systems for insect cells, or (my personal favourite) the rabbit reticulocyte in vitro expression system—are worth every last grant-awarded penny. You have to make the judgement call, and you need to learn how to do that. You have that responsibility.
It’s also because some of the reagents that you get in kits are more dangerous than the stuff you’d use if you did things the traditional way (name the contents of the first wash buffer in the Qiagen miniprep kit…).
And on a lighter note, I used to roll my own weblog. I knew a ‘kit’ existed, but didn’t use it; until it suddenly just got too much and I switched to MT4 WordPress. Talking of which, Bill Hanage is now blogging for LabLit. Go read.
In light of certain threads on the bloggers’ private forum, perhaps you want to modify a couple things in here? Starting with balk? 😉 No, but really, perhaps l’air du temps is caution?
Balk? Eh?
Dunno Heather. Strikes me if you can’t make statements of fact then, well, sod it.
I happen to like plasmid prep kits, if only because if I really really need something right away, they’re pretty fast; I don’t have to wait for an alcohol precipitation, I don’t have to spin for a phenol chloroform extraction, I don’t have to air-dry my pellet. Even if there’s more hands-on time, I can go from culture to sequence-grade DNA in 30 mins. Same thing with the maxipreps, now that our lab uses the CompactPrep kits with the syringe filters. Sure, we can’t overload the columns, and yes, it’s pricier than home-grown kits, but frankly, $1 / miniprep is just a drop in the bucket.
I can go from culture to sequence-grade DNA in 30 mins
That’s slow. I can do it in ten.
@Eric $1 / miniprep is just a drop in the bucket.
as a one-off procedure I would agree but when you have, say, 6 students cloning and they are going through a box of 250 per week (I kid you not) and you are paying AUD700 per kit then it adds up significantly to the budget. Teach the students what is in each solution, what happens at each step [usually] means they can trouble-shoot if they need to and not i) waste even more columns and ii) not waste my time by saying “but I followed the instructions and it still didn’t work”. Um, clearly you didn’t…
I should add that’s 10 minutes for the kit, not the homemade method (don’t diss the importance of measuring hands-on time by the way. Multitasking is essential for today’s scientist).
And what I didn’t rant about was the lack of innovation in this field. Blue dye, that’s about it. For part of the process that really, isn’t that difficult. Where are the long-hinged collection tubes (I put those in a kit in 1998)? And did you know that to get your best yields, air-drying the membrane and using 60°C water/TE is the win? See what understanding does for you?
This post and comments have me realize just how much there is to know about every detail. I thought I had put in some effort in figuring out what the kits did but clearly I have a lot to learn.
On the topic of precast gels – what about ready-made 7 min western blot transfer apparatus? I haven’t made up my mind about that one yet – when we first got it in the lab I was set against it and wouldn’t let it touch my proteins, but I am slowly being won over. 7 minutes! No methanol! But it IS a waste of money.
I would be very tempted by that, Anna.
You see, you have to ask yourself, is it a waste of money?
Seven minutes from gel to blot? Sounds great. But basically that’s seven minutes in which you can’t do anything else, really–whereas with the old fashioned method you could have done 10 sets of minipreps. But then again, maybe you get perfect results each time (no bubbles? I’d like to see that), as well as getting more Westerns done in a day/week/career. And no methanol is a bonus, too.
Thing is, your time is money and sometimes it’s worth it. What I’m trying to say is you have to judge each case on its merits. I’m not saying ‘all kits are bad’. Some kits are actually bloody good.
Jenny would say that you don’t have to understand every detail, I think. And sure, at first pass you don’t. But when things go wrong (which they do) or you want to optimize/change a procedure (which you will) then it makes sense to understand what’s going on.
I believe I actually said that you don’t have to understand the details unless it goes wrong.
Could be. laughs
I think it helps to know a little of what’s happening though, even when they don’t. Or maybe that’s just me: I don’t like black boxes.
But when things go wrong (which they do) or you want to optimize/change a procedure (which you will) then it makes sense to understand what’s going on
I learned the hard way… I worked with microarrays, so imagine that everything is prepared in little and cute kits: DNA isolation, DNA cleaning-up, etc, etc… you name it, I’ve done it!…
It took me a long time to understand every kit and what was going on there, because of course, things didn’t work the way I wanted them to… (and I’m also the kind of person that does not like black boxes).
Haha. Actually, when I did my exon microarrays, I prepped the RNA and actually (tiny letters)outsourced. I gave the RNA to a friendly scientist at the Ramaciotti centre who sent me the results a week later. Now that’s black-boxing, but seriously, it saved me so much heartache.
Hmm. Technicians as kits. There’s a blog post.
Heh. Our microarray facility (the Affymetrix part, anyway… Agilent and Illumina being totally different animals) has built their business on tweaking (with concomitant understanding) the protocols, most of which as María says are kit-based. You can get into a protracted discussion with them about the exact number of seconds for the DNAse fragmentation step, but I wouldn’t recommend it unless you have some time to kill while waiting for the bus to the station.
I am of two minds regarding plasmid kits… I did buckets of my own-brew preps which were the business, but when Qiagen (well, I think Stratagene showed up first in our lab, or maybe it was somebody else – horrible little column thingies that sat in the tops of Eppies) showed up, I was on board. Ease of use and all that. But I was lucky enough to be in well-funded labs, and competent enough (which isn’t saying much really) not to, as Kate says, screw up by mis-reading the instructions.
I will refrain from rambling on about S-35 sequencing. Total agreement that it was a great skill to be an ace at, and also now completely irrelevant and definitely not something I’d wish to re-visit.
@RW S-35 sequencing
Definitely much easier to get sequencing now outsourced but I do have fond memories of sitting on the green in Oxford outside our department holding the autorad up to the sky to read the sequence…
@RW S-35 sequencing
Note to self: preview comment before submitting – that should read ‘easier to get sequencing outsourced now’
Don’t talk to me about DNA fragmentation… I remember a postdoc when I was a grad student doing precisely-timed sonications for cloning.
Oh. Another thought here. Has anyone looked at how much plastic is in these kits and compared with non-kit methods? Should also compare reagent use for maximum green creds.
Not to mention the inevitable bucketload of “buffer Q” or whatever that you get left with, because the company just doesn’t know how their own protocol works.
@Kate – I did like reading those gels, and the “flip it over and spin it end-to-end” trick to read the reverse complement is a true thing of beauty. Beats all heck out of pasting a sequence into some website and hitting “go”.
@RPG – timed partial digests for pulsed-field gels. It is teh evil.
I did quick-and-dirty plasmid preps from scratch, skipping the phenol/chloroform step completely. It was easily good enough to see if you had the right sized insert, and I’d then clean the likely looking plasmids up for sequencing using a kit. A good approach, I thought!
I once told an Ambion rep that their 5′-RACE kit saved my project. It really did – the home made method was completely resistant to trouble-shooting, and after 3 months of failure, I begged my PI to be allowed to try a kit. She agreed, and I had the much-needed result within 3 days.
I left betaGal doing RACE in Sydney.
With a kit. I might be grumpy, but I’m not cruel.
I did quick-and-dirty plasmid preps from scratch, skipping the phenol/chloroform step completely.
Cath, is that the ‘fish out blob with toothpick’ method or the easy one.
It’s actually how I used to do them, without P/Cl, routinely: if I wanted sequence I’d tend to throw in some RNase (from a kit…) at the alkaline lysis step and do a PCl after isopropanol. Seemed to work.
One of the great things about DNA preps is seeing your DNA precipitate in ethanol. I think that’s the real reason the midi/maxiprep kits are so utterly terrible: the R&D guys know people like that step best so aren’t ever going to get rid of it.
The easy one, although my friend always tried to get me to switch to his toothpick method. The fact that he called the blob “snot” did not help his cause.
No…
I can’t imagine doing 40 blob method ones at a time (which I used to do with the ‘easy’ protocol).
@Richard Do you just shorten the genomic-DNA-precipitation spin to get under 10 mins (seeing as that step is 10 mins in and of itself)? I’ve never bothered fiddling with the steps, because my miniprep haven’t ever been troublesome, but a savings of time would not be unwelcome.
Every spin step except that one can be done in 15s. That one, two minutes: which gives you time to label the tubes for the next step.
And I’d seriously recommend leaving the baskets to dry for (coffee time) and eluting with 60°C water/TE. If you can spare the time, of course…
Oh, and PS: don’t ever, ever, ever incubate in NaOH/SDS or KOAc. The lysis is +/- instantaneous; neutralization doubly so.
Handy. I’ll try it next time! Generally, cloning is one of those things I’d rather just power through to get to my real experiments, hence my preference for things with shorter time overall.
And of course, you know about colony PCR, don’t you…?
Agree with RPG (it happens, sometimes). Qiagen kits require no incubation steps and the spins can be just as long as it takes the ‘fuge to get up to speed and down again.
Saved me hours, hours I say.
Getting back to home-brew, people in this general vicinity used to use some rapid boiling prep to make sequencing-grade miniprep DNA. Fabulous.
baulk (British) Alternative spelling of balk. (I didn’t know.)
Statements of fact are all very well until the shit minipreps hit the fan, no? Ask Simon or Jack about it sometime.
I’m a fan of the rapid boiling lysis for miniprep DNA personally, but I don’t think it’s sequencing-grade. It’s enzyme-digestible, though. It’s in reality about an hour from start to gel, and most of that is liquid transfer. I also love the way the blob of other crud adheres to wood toothpicks, so wouldn’t pass
that up (EtOH precipitation is wonderful though on dry ice, if you happen to have any on hand).
My dictionary says ‘balk’ is the alternative. But hey, that’s a silly argument.
Doesn’t boiling nick your DNA? Am quite intrigued about the chemistry, actually.
Hm. Nick it chemically, or simply mechanically as it gets agitated in solution?
I’m interested too.
To be honest, I can’t remember the method so I’m making this shit up.
I do know that the standard alkaline lysis kit method nicks yer DNA, if you RTFM. At least, it used to. Have they changed it now?
Pfft. As you well know, you’re asking the wrong person.
Well fetch me the right one, dammit.
Have your fellow Torontean here. I’ll go ask her.
“Torontonian”.
I bet she didn’t know either.
More like ‘Trontian’.
“I bet she didn’t know either.”
I did know that. I also know what people from Halifax, Liverpool, or Sydney are called, and I’ve never even lived in any of those cities!
What about people from Glasgow and Newcastle?
What about them?
Does Eva know what they’re called?
RG> What they are called…. I have some kind of recollection of “Scots with nonunderstandable accents when they been home for Christmas break” and “people from the place with good ale”. .. .then again, it feels like I might have misunderstood that one 😉
[I had more rude memories but decided to leave that for now…. ]
Uhm….Glaswegian…and…don’t know Newcastle.
Colloquially, Geordies. Properly, Novocastrians. But well done on the Weegies, and the Haligonians!
Cath: I thought about Geordies (learned it from Ian Rankin and his Rebus books) but somehow I get this “knives” images up in my head too 😉
I did know that
I meant the thing about DNA nicking. Whatever. How’s your hangover?
Don’t ask her, she’s on a plane somewhere over Greenland.