Life Science Tools of the Trade

In researching (and I use the term in its modern sense, roughly meaning “looking on the internet for approximately three minutes) in order to find something to contribute to a discussion about inappropriate scientific article titles, I came across this absolute gem of a paper:

All’s Well That Ends Well: Shakespeare’s treatment of anal fistula. Cosman BC. Dis Colon Rectum. 1998 Jul;41(7):914-24. PMID 9678380

I had no idea, really, although I suppose that in a journal called ’Diseases of the Colon and Rectum’, I shouldn’t be surprised. With morbid fascination, I read the abstract:

Textual and contextual evidence suggests that the French king’s fistula, a central plot device in Shakespeare’s play All’s Well That Ends Well, is a fistula-in-ano.

Really? I’m fairly certain I’ve seen this play performed, at least in a television adaptation. It was a long time ago, and certainly long before I began to be interested in gastrointestinal disorders, but I would have thought I would remember references to the French king’s backside. And I’ve already learned something else:  I don’t think I’ve ever come across the term ‘fistula-in-ano’ before.

Reading on:

Anal fistula was known to the lay public in Shakespeare’s time.

I suppose that makes sense. I hadn’t really thought about it.

In addition, Shakespeare may have known of the anal fistula treatise of John Arderne, an ancestor on Shakespeare’s mother’s side. Shakespeare’s use of anal fistula differs from all previous versions of the story, which first appeared in Boccaccio’s Decameron and from its possible historical antecedent, the fistula of Charles V of France.

Ok, now the author’s getting serious. Or the article’s getting silly. One or the other. Onward:

This difference makes sense given the conventions of Elizabethan comedy, which included anal humor.

Again, I hadn’t thought about it - but no surprise there, really.

It is also understandable when one looks at what wounds in different locations mean in European legend. In this light, it is not surprising that subsequent expurgations treat Boccaccio’s and Shakespeare’s fistulas differently, censoring only Shakespeare’s.

Well, ok - they removed the reference to the King’s bum. I’m not really surprised. Other ‘wounds’ were much more socially acceptable, I suppose. After all, we’re talking about a culture that endorsed public beheadings.

Cosman’s abstract ends with this screechingly funny statement:

This reading has implications for the staging of All’s Well That Ends Well, and for our view of the place of anal fistulas in cultural history.

Indeed it does. I shall never view the cultural history of anal fistulas in the same way again. Or perhaps at all. And I’m certainly going to be paying closer attention the next time I see All’s Well That Ends Well performed. Paying closer attention, but ready to run for the door if the King’s nether ailments are about to be revealed.

It’s a good thing there are curated literature search engines, like PubMed. Otherwise, how would I ever find these things?

I happily confess that I haven’t read many of Charles Darwin’s published works. Of those few that I have, one of my favourite passages (from The Voyage of the Beagle) is this:

“In the evening we reached the island of San Pedro, where we found the Beagle at anchor. In doubling the point, two of the officers landed to take a round of angles with the theodolite. A fox (Canis fulvipes), of a kind said to be peculiar to the island, and very rare in it, and which is a new species, was sitting on the rocks. He was so intently absorbed in watching the work of the officers, that I was able, by quietly walking up behind, to knock him on the head with my geological hammer. This fox, more curious or more scientific, but less wise, than the generality of his brethren, is now mounted in the museum of the Zoological Society.”

Poor old fox. Little did he know his place in history.

Darwin, I suppose, was simply following accepted Natural History custom of his day, collecting a specimen for the museum back home. I know people who do this now, embarking on “prospecting” missions to exotic locations, to collect, catalogue, preserve, and, yes, bonk on the head (or the entomological equivalent, drop in alcohol) various unfortunate creatures. Perhaps even threatened or rare ones - by mistake, one might hope, or through ignorance of their scarcity. I recognize that Darwin’s time was different than today. But even so, it grates on me a little that he ”collected” the trusting Canis fulvipes quite so cavalierly.

Ironically, we can perhaps stretch the blame to encompass Darwin’s nature: in his autobiography, he notes that “The passion for collecting which leads a man to be a systematic naturalist, a virtuoso, or a miser, was very strong in me, and was clearly innate, as none of my sisters or brother ever had this taste.”  But perhaps that’s playing the deterministic card a bit too much. And after so many years, Darwin hardly needs the likes of me to cut him some slack.

In researching this piece, I came across this rather endearing illustration of the little fox, who it appears now goes by the altogether more exciting name of Pseudalopex fulvipes. You can read all about him in this paper by J. E. Jiménez, or on this website. Unfortunately, it seems as though time hasn’t treated Darwin’s fox well, and he and his vulpine friends are still critically endangered.

Which brings me, via a tortuous route, to the point of this piece - the hammer. As a scientific tool, it’s better suited to the geology for which Darwin brought it along, than the Natural History to which he applied it on this occasion. But regardless of method, the result is that Canis fulvipes ended up in a museum collection, to ultimately be re-classified in the genus Pseudalopex (or perhaps Lycalopex - I’ve become rather confused in trying to sort this out). I confess I haven’t looked into the history of when and how this was done - whether on morphological, or genetic grounds, or perhaps a combination of both. But I can’t help wondering what Darwin would have made of the modern tools we have to aid us in classification in the Twenty-First Century -  this whole new discipline called “Phylogenetics“. Nurture may be one thing, but Nature leaves a fingerprint, and if you have the right skills, you can sort the evolutionary relationships out using a molecular hammer of much smaller size, and much larger impact.

None of this is news to anybody, I suppose - but imagine, if you will, what Charles D. would have done with a modern-day arsenal of analytical tools at his disposal. Think of the books he would have written. And then go and look at the books he did write, and be amazed. With little more than a notebook, a keen set of observational skills, and that all-important hammer, Darwin changed the way we all look at species, their relationships, and their evolution. And he was even stealthy enough to strike down poor Pseudalopex while it was distracted.

Happy Darwin Day, everyone.

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Some further reading, of the technical sort:

J. E. Jiménez  (2007). Ecology of a coastal population of the critically endangered Darwin’s fox (Pseudalopex fulvipes) on Chiloé Island, southern Chile. Journal of Zoology, Volume 271 Issue 1, Pages 63-77. Jiménez and co-authors have written a number of papers about this species.

B. Rannala and Z. Yang (2008). Phylogenetic inference using whole genomes. Annual Reviews of Genomics and Human Genetics, Volume 9, Pages 217-231. PMID 18767964 A technical, but not completely overwhelming, treatment of a very complex subject.

Following on from my last post, it appears that the Canadian federal government has decided not to fund Genome Canada at all. Which is dreadful news for the research community that I inhabit.

You can read an article by reporter Carolyn Abraham that made the front page of this morning’s Globe and Mail, right here (and excerpted below).

For the first time in nine years, Genome Canada, a non-profit non-governmental funding organization, was not mentioned in the federal budget and saw its annual cash injection from Ottawa - $140-million last year - disappear.

“We got nothing, nothing, and we don’t know why,” said a stunned Martin Godbout, Genome Canada president and CEO. “We’re devastated.”

This is potentially going to make it a lot harder to attract and retain highly qualified personnel to run these fancy “tools of the trade” that we have in our labs. There’s one last recourse I can think of - I believe the final debate on the budget is today. We’ll see if anything changes. Until then, I’m going to mope.

“Tools of the Trade”, you say. Well, I’m going to bend the definition of this blog a little (again), this time towards funding. Which is, of course, what enables us all to purchase the various and assorted goodies that we like to put in our labs.

Connection made. I’ll move on now.

For those of us in Canada, next Tuesday will be a day to watch. While our neighbours to the south have been wrapping up the hoopla surrounding the inauguration of their new President, we in the north have been quietly creeping closer to the tabling of a new federal budget. Our Minister of Finance, who tradition dictates I must refer to as “The Honourable Jim Flaherty“, will be dropping this particular bomb on us on the 27th.

This impacts many things, but the one I’ll be watching most closely is the funding allocation for Genome Canada, which is not only the major funder of large-scale “omics” research in this country, but also distinctly not a persistent line item in the federal budget. Each year, they lobby for funds based on the competitions they envision running and ongoing initiatives they need to support, and each year we all wait with varying degrees of bated breath to see what happens. Early in 2009, our federal administration is pretty shaky indeed, having been prorogued over the Christmas break, essentially to avoid a non-confidence vote headed by a coalition of opposition parties. Ah, the joys of a minority government. While things seems a bit more stable now, a mis-step on Tuesday could nevertheless bring the government down.

So, we’ll wait and see. I had, but did not take, the opportunity to introduce myself to the Minister while he and I were the same flight back from Washington in November (he in first class, returning from the economic summit, me in economy, returning from having lunch with a couple of fellow SAB members). Perhaps I should have mentioned genomics research to him then… but maybe it’s best I left well enough alone.

See you on Wednesday, then.

I’ve shamelessly plagiarized this from my personal blog. I guess I’ll have to pay myself royalties, or something.

For those of you who enjoy blogging, this looks like it could be fun:

It’s presented as a “blog swarm“, where not only are the organizers soliciting Darwin-themed blog posts to celebrate the bicentenary of Charles Darwin’s birth (February 12th, 1809), but are intending to aggregate them all as well. Regardless of your views on Darwin himself, his scientific prowess (or not), his writings, or evolution vs. intelligent design vs. whatever else, this still sounds like a very interesting initiative. As I’m firmly in the Darwin camp (although with reservations about certain of his published works, some of which are very tedious indeed), I’ll be participating, both here and over at the aforementioned personal blog.

You can too… click right here to find out how.

Thanks to Heather for alerting me to this.

Considering that I’ve managed only seven posts since my first one last March, and my fellow LSTOTT bloggers hwiegand and hbogerd haven’t posted in nearly four months and seven months, respectively, I’d say that this place is not exactly staying topical and up-to-date. In fact, it’s barely ticking over.

And, given that the average number of comments over the last ten posts is just less than two (1.9, range 0 - 5), with quite a few of those being my own responses to initial comments from others, I’d also venture to suggest that barely anyone’s reading it. Either that, or those who do are remarkably silent.

So - this begs the question. Is there any point to continuing this blog? I suppose it’s gratifying to see comments, and feel that others are reading, perhaps being stimulated to think about the topics of the posts a little, or at least being engaged. I know that the argument’s been made many times, by many people, that “we don’t blog just to get comments”, and I guess in some way that’s true. But I, personally, don’t blog in order to simply throw my thoughts into the wind - if nobody’s reading, what’s the point of writing publicly? I might as well keep a little notebook, or put it all on a private website.

This all may seem like whining, and perhaps it is. But if anyone’s interested in commenting, I’d like to hear it, as long as you’re not the same people who’ve been hitting the blog with spam comments about various activities consenting adults might engage in. Oh, and yes, I’m aware that I’m not the only SAB blogger who’s been thinking about moving on, shutting down, or finding another forum for his/her thoughts.

We’ll see, I guess. Happy New Year, everyone - new things, and all.

…we’ve also installed one of these:

Yes, that’s a Roche GS-FLX, which will be fully loaded with their latest “Titanium” upgrade. Actually, it might already have it - I confess I’d have to ask someone in the lab, or maybe the office next door. Joining the other instruments from Illumina and Applied Biosystems, and promising greater than 400-base paired-end reads, this should solve a lot of (biological) problems. We hope.

But - are we behind the curve, again? Pacific Biosciences has announced that they are gearing up for an early-access program in the first half of 2009 (and I apologize if you can’t see that article, which might be subscription-only), meaning that they might actually have functional instruments on the market in 2010 as they’ve been saying since February. If this thing actually works as advertised, it will be game-changing in the extreme, and will make the other three competing instruments look stodgy, expensive, and slow.

PacBio has thoughtfully provided a nice little video on their website for you, if you’re interested in this technology. Warning - the term “zero-mode waveguide” is used, although the rest of the description is very basic. The claim is made that entire genomes (or, presumably, genome-sized complex template mixtures, like transcriptomes) will be sequenced in under an hour, at a cost of hundreds of dollars. If true, this will just about spell the end of the microarray for most applications.

We’ll see, I guess. Applied Biosystems certainly has other, competing technologies in the pipeline, but I doubt very much that Illumina has the resources, or Roche has the vision, to stay competitive in the long run. As for Helicos, well, I’ve changed my previous optimistic tone and have switched to predicting their demise for a while now, although they recently bravely reported that they will sell five to ten instruments by the end of the year. I’m not convinced, but I guess we’ll see. In the meantime, the Illumina, Roche and AB instruments appear to be working now, and our three-pronged, high-throughput attack on biology is continuing unabated.

Interesting times.

If there’s one thing my PhD supervisor taught me (and I’m hoping she taught me more than just this, but this one is important):

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“Data” is a plural noun. Data are plural, they are analyzed (or analysed, if you prefer) and when you post them to that website, they HAVE been saved.

Thank you for your attention.

One of the pipette companies had a *mini* vendor show here the other day. As everyone probably knows, I don’t think very highly of pipette calibration companies. This company, however, also sells different brands of new pipettes as well as refurbished ones.

Since we have way too many pipettes in the lab to replace them all with new ones, I’m thinking about getting some refurbished ones. The price is a lot less than new pipettes and the refurbished pipettes have a 90 day guarantee.

I figure I’ll try one new set and see how it goes.

I’ve spent considerable amounts of time here, elsewhere on the Science Advisory Board, and in a few other places, writing about so-called “next-generation” sequencing. While some vendors are already shying away from this “next-generation” moniker, it’s a good enough catch-all phrase for now, I think. Anyway, most people seem to know what it means.

The currently available technologies include the Illumina (née Solexa) Genome Analyzer II (replacing the flaky Genome Analyzer), the Roche (née 454) GS-FLX (replacing the original GS20), and the Applied Biosystems SOLiD (still in its initial release). There’s also the Helicos HeliScope, which as far as I can tell is kind of like the mythical white elephant, or the Loch Ness Monster - many agree that it exists, but nobody’s ever seen one.*

Jumping in to this game rather cautiously, we ran a very unreliable GA for a while, and after considerable beating up on it have obtained some very nice data (in particular, for some ChIP-seq experiments, where the miniscule 30-ish base reads from this instrument are ideal). It’s now in the process of being replaced by the GAII, and for the time being both are co-existing on the same bench.

Spot the difference.

But, not to fall prey to the single technology trap, we’ve also added an Applied Biosystems SOLiD, which is currently being installed. Here it is with its doors open (look away, children).

This sequencer photo is rated PG-13 for partial nudity.

And, just to make sure that our bases are completely covered, we’re also ordering a GS-FLX, since it seems there will be quite a lot of genome-sequence backfilling in our future, and the long reads (with reliable paired-end capability) should do the trick.

Fun and games. Our old 3730xl capillary sequencers would be feeling sad, if they weren’t so darned busy screening for mutations and making sure everybody’s plasmids are what they’re supposed to be.

A few years ago, this NGS stuff all seemed like voodoo. Now it’s here, and, like any good scientists, we’re itching to play with it. Good times.

*I have shamelessly stolen this analogy from a colleague. It can equally well be applied to the results of certain experiments that are supposed to have been done around here.

The lab budget and the books are my boss’s job. I just try to keep the lab stocked and the supplies ordered for the lab. That said, I shop around a bit and buy when items are on sale. Most of our lab supplies have stayed pretty constant through the years. Restriction enzymes, tissue culture supplies, chemicals, the prices on these *old* lab items haven’t changed much and they’re not that expensive.

What I am noticing is the expensive price of all the *new* supplies. The RT-PCR reagents, the deep sequencing supplies, the modified oligos, FACS analysis… those items are pricey! The control for the RT is a couple hundred. The oligos a few hundred. Is it the new technology or the times dictating the prices? I’m not sure. It does make me wonder though how some labs can afford to do these experiments. I’m assuming the price will eventually come down on these items. In the mean time, I wonder how many experiments aren’t performed because the cost of doing them is just too high for the lab.

I hate phones. I don’t have a cell phone. I let the answering machine pick up 99% of calls at home. At work I answer the phone. Not because I’m looking for another annoying cold call from Dipshit Biotech but because I have a wife and two kids there are times I need to be reached. Plans change, illness, car problems, etc. So……….

Today the phone rings in the lab. I’m in the middle of setting up a fairly complicated experiment. “Hi Hal, I’m ____________, you probably don’t remember me but you sent me some reagents several years ago and I want to do another experiment and wonder blah blah freaking blah.”  Short of hanging up (why didn’t I think of that), how the hell can I escape? Ten minutes later I’m back at the bench, dazed and confused. Did I finish the master mix. Start over…………..not enough left of one reagent………..I find more but it is another prep…recalulculation time……….feelings of failure…finally I toss the whole thing in the trash can……….”That bitch”….ooops, that costs me a quarter to the lab curse jar.

But it was worth it. Money well spent.

Tomorrow?

 ”One more chance to get it all wrong”

(lyrics courtesy of the Replacements)

So, hearty congratulations to fellow LSTOTT blogger hwiegand, on the topic of her latest post (oh, go on, scroll down and have a look). I suppose that’s an excuse for not blogging for a while. Like I should be talking.

As usual, things are very, very busy around here. Why, today alone I had to have coffee with a representative of a local software company (might be a consulting opportunity there, who knows?), followed after a respectable break to do a modest amount of work by lunch, in the presence of representatives of a company that would be happy to sell you a great big, hefty, chunky (dare I say “solid”?) DNA sequencing instrument.

Ah, the client lunch. Just what one needs on a lazy Friday at the tail end of an already holiday-shortened week. Did I say “lazy”? Perhaps I mis-spoke.

Said lunch was made slightly more amusing by us all bumping into a senior-ish person from an interesting company that would be equally happy to sell you a brilliant, shiny (dare I say “illuminating”?) DNA sequencing instrument. I ducked; my colleague stood by, smiling happily, as if a brawl worth watching were about to develop. Civility prevailed, and we all went back to work. For another short stint, just long enough to assuage everyone’s consciences before heading home for the weekend.

Which leaves me with a whole lot of follow-up to do on things that have been sliding. But all flippancy aside, these kinds of meetings with industry personnel, whether sales and marketing, technical, or even executives, can sometimes be very useful. They can lead to collaborations, discounts and deals, insights into future directions, even an invitation to an event or meeting can fall out of them. Like it or not, this is part of my job. I see it as an essential way of distracting the company folk from bothering the people in the lab, who really do have better things to occupy their time.

How’s that for justification?

I apologize for my lack of writing the last month. April passed by in a blur for me with final touches and preparations for my wedding followed by two weeks in Italy on a honeymoon. Needless to say that’s ALL that I accomplished for the month. BUT what an accomplishment!!

The wedding was great! We celebrated with family and friends and were married at the end of the day. The following day we were off to Italy - a first time visit for both of us. A few days in Venice, a few days in Florence, and to finish off the trip a few days in Rome. Not a lot of science. Actually there wasn’t any science but there was a lot of history, art, good food and good wine.

A wonderfully memorable and exhausting trip!!

So, a local sales rep for a certain Qompany that sells mainly kits, but also robots, came by my office recently with a promotional flyer. An invitation, if you will, to a V.I.P. event - a showcase of their new robotic, liquid-handling, sample-preparing, all-singing, all-dancing box. A box with a lovely name, evocative of orchestras.

Now, I’ve made no bones in the past about not this not being my favourite of companies, their sales tactics ranging from dubious to downright annoying. And this fits right in… a V.I.P. event, you say? Then why, I ask, is it from 9:00 AM to 4:00 PM in a conference room? With refreshments all day? “Oh, don’t post this notice, I don’t want this open to everyone… just a select few.” They sure know how to make a fella feel special, I tells ya.

Oh, and the robot in question? Well, the sales rep described it to me as being a box that you can put any kind of sample into, and get any kind of prep out. Cells in, RNA out. Brain in, DNA out. Fossilized pterodactyl bones in, highly purified Jak Kinase out, that kind of thing.

I think I may give this one a miss.

In this post-90’s biotech era which we all inhabit, I’m finding that brand recognition is becoming a confusing game indeed. Old favourite boutique vendors like Molecular Probes have been swallowed up by enormous, multi-tentacled distributors like Invitrogen, and keeping track of who’s distributing your favourite brand of pipettor, or water filter, or tissue culture media, can make for hours of fun and games. Even the big players keep getting bought and sold – just try to sort out the whole Merck/EMD fine chemical business, or the ownership structure of VWR, if you have some time to kill. And I’m still trying to get my head around Thermo Fisher. What does Thermo Electron have to do with distributing pipette tips and latex gloves? How, if at all, is this related to Thermo Finnigan? It makes my head hurt just thinking about it.

And then there’s my personal favourite suite of technologies du jour, loosely grouped into “next-generation” DNA sequencing, or NGS. Illumina buys Solexa, Applied Biosystems buys Agencourt Personal Genomics (but not Agenourt Bioscience, which is owned by Beckman Coulter – are you following this?), Roche gobbles up 454 Life Sciences. Pacific Biosciences is next, mark my words, with rumours of intense interest from Applied Biosystems, and probably many others. Helicos too, perhaps, so look for a merger or acquisition there, although with a market cap of $147 million and $50 million in the bank at the end of 2007, they could probably stay on their own for a while.

In the case of Illumina’s almost-works-most-of-the-time Genome Analyzer, most people still call it a “Solexa”. At the recent AGBT conference, which I’ve rattled on about in more detail elsewhere, practically the only people using the term “Illumina Genome Analyzer” were members of the large posse of Illumina employees in attendance. And most people don’t call the ex-454 machine a “Roche” GS-FLX; to most, it’s still a “454”, although this seems to me to be waning a bit under the crushing weight of Roche’s marketing machinery. Remarkably, the 454 Life Sciences website still exists, and is still a much, much better source of information on this NGS system than the Roche website, which is large, messy, and rather full of the 150,000 other things that Roche sells.

On the other hand, Applied Biosystems seems to have triumphed in branding their SOLiD system, and virtually nobody seems to remember that this was developed by Agencourt Personal Genomics and that the chemistry used was, for a time, referred to as the “APG process”, even by AB itself. Now it’s just SOLiD, small “i” and all, and the scientific community in general seems to have accepted that brand. Timing, I suppose, is everything.

Now, if someone could just explain to me why all these darn NGS boxes are blue…

The latest email from them says this is my third and final notice. If I do not respond to this email they will terminate my subscription. Thank goodness.

This is one of those mailing lists I got on from a vendor show and don’t know how to get off of. I have my doubts whether not responding to the email will actually terminate the subscription but I’m keeping my fingers crossed.

These types of magazines might interest some but I don’t have any interest in thumbing through a magazine of advertisements.

Well, as Kat mentioned over at the SAB, I am now activated, Word-Press-enabled, and ready to jump into this whole Life Science Tools of the Trade thing, joining my virtual colleagues hbogerd and hwiegand.

As some of you know, I’m a Blogger kind of person so far, so this post is as much about introducing myself as it is about seeing whether I can work out how WordPress works. So far, so good, nothing’s caught fire yet. I will doubtless be posting many erudite comments on the technologies we all use (or more likely, those that fail us miserably), but in the meantime I’l leave you with a teaser: yes, we have a next-generation DNA sequencer; no, it doesn’t work as well as advertised; and we just finished our first client project, a ChIP-sequencing experiment, after seven months of trouble-shooting since delivery. That’s about average, folks.

At a vendor fair I picked up a free sample of Agarose Tablets from BIOLINE! The little white pills of agarose eliminate the  messy task of weighing out agarose! Okay, unlike LB powder or SDS, weighing out agarose isn’t really that messy. The little pills do save you the time you’d spend weighing out the agarose. Unfortunately, the pills have to dissolve for for 6 minutes in your buffer before you can microwave/melt them. Okay, no time saved. Safer to use?? I have no idea what that even means. The agarose tablets are probably MORE dangerous. Swallow one of those suckers by accident and I bet you’d be plugged up for days. Oh yeah, and they’re more expensive.

Thanks but no thanks.

A co-worker asked if I’d help her with some cloning. She had the strategy all figured out. The oligos were here and the pcr was done for the insert. All I had to do was digest the insert fragment and the vector backbone, purify them and ligate them together. Seemed like it should be easy enough.

That was until I looked at the enzymes. Urgh! They were two of those enzymes you only use under dire conditions. One cuts at 37 degrees, the other at 50. AscI only cuts 100% in NEB buffer 4, the other one only in buffer 3. I gave her a hard time about passing the more *difficult* cloning on to me. She said she knew about the temperature difference but that both enzymes cut in buffer 4. Huh? I had just checked the NEB catalog and knew that wasn’t right. BsaI only says it has 50% activity in buffer 4.

What was going on? We both felt like were going crazy. She brings her 2005-2006 NEB catalog over and shows me. BsaI and AscI both cut in buffer 4 - 100%. I look in my 2007-2008 NEB catalog and BsaI only cuts 50% in buffer 4. Turns out were both right. Sorta.

I’m not sure what changed. Is NEB purifying the enzyme differently? Did they just realize it didn’t cut as well as they had thought? Whatever the reason, the recommend buffer has changed. I’m not sure how many other buffers have changed but if you have an old NEB catalog hanging around you might what to get a newer one!

PS - Turns out neither one of us has had a whole lot of success with the cloning so far. I’m still not sure which is the best buffer to use!