{"id":292,"date":"2009-08-17T19:32:00","date_gmt":"2009-08-17T19:32:00","guid":{"rendered":"http:\/\/occamstypewriter.org\/rpg\/2009\/08\/17\/on_kit_culture\/"},"modified":"2009-08-17T19:32:00","modified_gmt":"2009-08-17T19:32:00","slug":"on_kit_culture","status":"publish","type":"post","link":"https:\/\/occamstypewriter.org\/rpg\/2009\/08\/17\/on_kit_culture\/","title":{"rendered":"On kit culture"},"content":{"rendered":"

Jenny’s post last week<\/a> 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-\u00e0-vis pre-cast gels.<\/p>\n

\nFirst, some history.<\/p>\n

\nBack 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<\/sup> 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.<\/p>\n

\nI remember my first ever PCR. I spent a week preparing\u2014under DNA-free conditions\u2014all 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 35<\/sup>S 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<\/a>.<\/p>\n

\nThe restriction buffers, the magic 10 X PCR buffer (and even the pre-made MgCl2<\/sub>) stock; yay verily the Sequenase kits: all these supplanted homemade methods and reagents in their time. Overall, a good thing, I say.<\/p>\n

\nAnd 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<\/em> wasn’t nanometer perfect; or who has found bubbles right at the resolving interfaces making half of all the gels unusable\u2014 anyone<\/em>, I say, would gladly spend a few extra quid on the pre-cast gels from Invitrogen. My boss in Cambridge actually instructed<\/em> 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.)<\/p>\n

\nBut there are\u2014or would be if I went back to the lab\u2014two kits that I baulk at.<\/p>\n

\nThe 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\u2014the cycling times and temperatures and advice on choosing primers\u2014and then bought Pfu<\/em> (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<\/em>. Minus a cardboard box.<\/p>\n

\n\"\"
\nMe, not seeing the point.<\/em><\/p>\n

\nThe other kit I hate (I say ‘kit’, but really it’s the whole industry) is plasmid prep kits. <\/p>\n

\nTake 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 \u00a31.09 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.<\/p>\n

\nGuess 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.)<\/p>\n

\nThe 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.<\/p>\n

\nSo 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<\/em> expensive.<\/p>\n

\nI say learn<\/em> 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).<\/p>\n

\nIt’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\u2014like Jenny’s bacmid kit, or those complicated expression systems for insect cells, or (my personal favourite) the rabbit reticulocyte in vitro expression system\u2014are 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.<\/p>\n

\nIt’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…).<\/p>\n

\nAnd on a lighter note, I used to roll my own weblog<\/a>. I knew a ‘kit’ existed, but didn’t use it; until it suddenly just got too much and I switched to MT4<\/del> WordPress. Talking of which, Bill Hanage is now blogging for LabLit<\/a>. Go read.<\/p>\n","protected":false},"excerpt":{"rendered":"

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 … Continue reading →<\/span><\/a><\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[1],"tags":[],"class_list":["post-292","post","type-post","status-publish","format-standard","hentry","category-uncategorized"],"_links":{"self":[{"href":"https:\/\/occamstypewriter.org\/rpg\/wp-json\/wp\/v2\/posts\/292","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/occamstypewriter.org\/rpg\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/occamstypewriter.org\/rpg\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/occamstypewriter.org\/rpg\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/occamstypewriter.org\/rpg\/wp-json\/wp\/v2\/comments?post=292"}],"version-history":[{"count":0,"href":"https:\/\/occamstypewriter.org\/rpg\/wp-json\/wp\/v2\/posts\/292\/revisions"}],"wp:attachment":[{"href":"https:\/\/occamstypewriter.org\/rpg\/wp-json\/wp\/v2\/media?parent=292"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/occamstypewriter.org\/rpg\/wp-json\/wp\/v2\/categories?post=292"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/occamstypewriter.org\/rpg\/wp-json\/wp\/v2\/tags?post=292"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}