Life Science Tools of the Trade

It looks like collectively the lab has grown tired with the ultra-pure fuzz contaminating every Qiagen DNA prep. Lately, Qiagen Maxipreps also gave a nice bonus of “insoluble crap” that transfected very poorly.
I tried the Roche kits and was pleased that the pellet was somewhat clear (DNA with some salt) and dissolved almost immediately.
I am always skeptical when someone tells me they bought a bad kit or reagent. In this case the entire lab had the same problem (Although historically there always was the “QiaFuzz” that the sales reps either pleaded ignorance about or said “Uh, I don’t know but I have heard about it”). As the problem worsened, my patience snapped and it is “Man overboard!” for Qiagen.

Music: Steve Earle “The Revolution Starts Now!”
Concert : Steve Earle solo at the NC Museum of Art
Science mood: in the hands of the reviewers
SAB mood o’ meter: excellent, but can it stay that way upon the return of G*******?

Due to the increasing fuzz problem from Qiagen maxi preps our lab has decided to make the jump to new kits and I think we’ve finally settled on the winner. We tried the eppendorf kit but after merely reading the instructions decided that kit wouldn’t work. Who wants a kit where the resin isn’t already in the column? The Sigma kit was all right but you need to elute your DNA in a fairly large volume. The kit ahead of the race right now is Roche’s. Their Genopure kits have given the best results with a small amount of change. The kit is essentially the same as Qiagen kits-alkaline lysis that you bind to the column and then elute. The major differences are: the Roche kits are CHEAPER and you DO NOT get any of the proverbial fuzz. After completing the Roche kit it was amazing to see the whole precipitated pellet resuspend.

The irony to the whole story is that we finally got a Qiagen rep that we would like to deal with but the product was lacking.

Nell brought doughnuts. If I eat another one I’ll enter a hyperglycaemic coma.

Note to self: don’t lick the jam and sugar from the tray.

I’m thinking particularly of K, who worked for BioRad when I was in Oxford. K was one of those rare (at the time) creatures who had a doctorate to go with their company car. Naturally she used to try to sell us BioRad gear, but she’d never lie to us if she thought another company did something better.

She understood the need to keep customers happy, and allowed us to trial a low pressure chromatography set up before the Department let me buy one, and naturally when I left that place I told all my friends (what’s that? Both of them? Yeah, hah bloody hah) about BioRad kit and tried to get them to buy it. K also didn’t wear strategic grade perfume and was very good at building rapport with the punters. I seem to remember going out to lunch down the Botley Road and - on K’s instigation - putting condoms over the end of the boss’s car exhaust. She went into the gents to get them, so I couldn’t really refuse.

I don’t remember why she left BioRad, probably getting ticked off at the way they treated her (and her customers). She went into business with some friends, selling stuff as a small, independent distributor. Got some nice things from them, including my favourite Nichimate stepper pipette and some very good low retention pipette tips (not coated; the beauty of these was that they were made from low retention plastic). She’s since moved on again, and I’m not sure where to. I should drop her an email and find out.

What companies seem to fail to grok is that reps are how people see the company. You get a good rep, one the punters like and get along with and can trust, and you get a good reputation. Reps like these, companies need to do all they can to keep them. And they don’t. Unfortunately the company and the punter lose out as a result.

Mood: green
Random musical fact: the opening chord of two riffs then an interval of a flattened fifth in Jimi Hendrix’s Purple Haze was a musical device condemned by the Spanish Inquisition.

I’ve ranted about Qiagen in the past. News Flash! It appears that we actually have a sales rep that gives a damn about us! Does that mean no more free movie tickets to a theatre 50 miles away to replace the $10 summer tote-bag that was back-ordered and we never got for ordering $10,000 worth of Maxi-Kits?
I wonder how many people are reading this blog after our mention in “The Scientist”? I’m still patiently waiting for the photo requests to come pouring in. Now we just need to fly the Caped Avenger to Durham for the group photo!
Oh, but back to science. I have been using Qiagen products for 10 years. What is that insoluble ball of crap that ends up in the bottom of your DNA after doing your $30 MaxiPrep? It must be some of the secret matrix that sloughs off during the elution step. It isn’t really a big deal when you get a nice yield from your prep (pcDNA for example) . Spin it down and throw away the fuzz and a bit of your DNA. However, if you are prepping some crap low-copy semi-toxic plasmid the “mystery fuzz” is a real inconvenience when you try and dissolve your miniscule amount of DNA in the fiberglass insoluble protective fuzz shield.

SAB thought: bummed about Nexins resigning
Book: “The Basic Eight” by Daniel Handler (AKA Lemony Snickett, yes that Lemony Snickett)
Music: “Rough Mix” Ronnie Lane and Pete Townshend ( a rare double purchase- vinyl and then cd)
Science Mood O’ Meter: “submitted” (cautiously optimistic)
Junk Food: Twizzlers
Recent analog to digital conversion: Frankie Yankovic’s “I Wish I Was Eighteen Again” (type-polka)

For those of you haven’t heard yet Qiagen is fool proofing their plasmid prep kits. We haven’t received the new kits in the lab yet but the rep has dropped off the literature. Apparently so many people were having problems doing a plasmid prep that Qiagen now has a color indicator in the buffers, LyseBlue as they like to refer to it. When you lyse your bacteria the solution turns blue and when you neutralize….it turns clear. I guess for some individuals they couldn’t tell that the solution got gooey and then the SDS precipitated giving white chunks.

Now I’m not saying that this color indicator is a bad idea. It never hurts for a company to idiot proof anything. In fact I really like the new color coding of the buffer bottles. I’ve managed to grab the wrong bottle and to try to lyse my bacteria before I’ve resuspended them! (I haven’t found a way of saving those samples.)

What concerns me are the individuals who can’t seem to get DNA from a kit. The rep has told me stories of support phone calls they receive and that they are hoping LyseBlue will solve some of these problems. I hope this solves these problems as well but if you’re in research and can’t master a plasmid prep….well…Good Luck!

Pretty content-free, a quickie if you like, but I thought the following article might be of interest to our reader: The opposite of eureka I: nurture — Mole 118 (16): 3567 — Journal of Cell Science.

The Mole makes an important point about testing reagents, and I must admit, this is something I’m very bad at. Maybe it’s something I should get good at, but often when you buy a kit there’s only enough reagent to do the experiments you want to do . . . it’s a courageous scientist who says

“I will ‘waste’ some of this kit to test that it’s OK.”

Surely that’s what (supplier-side) QA is all about? Yes, I know if we were working to ISO 9000 we’d probably have to do this anyway, but most of us aren’t. Most of us are ‘just in time’ scientists, and maybe we do need a sea change in our behaviour.

I need to go away and think about this.

Random science tip of the day: maxiprepped plasmid DNA is cleaner than a PCR prep. This is why the PCR prep ethanol pellet appears bigger than the plasmid ethanol pellet. Just saying.

Remember I wibbled about the IVGN stock fridge? Got a flyer in the post today from Promega. They’re introducing stock fridges with RFID scanning. The stock is tagged, so as you remove an item an order is automatically raised to restock.

This is a brilliant notion (not a new idea, I was planning a similar thing to keep my tonic stocked and cold at home ten years ago) but has potential pitfalls. Are multiple copies of the same stock item differently labelled (I can see someone in this lab deliberately waving the same item past the scanner so we end up with thirty thousand tubes of ligase)? How do we know who is taking the stuff? It’ll be a free for all, which is a killer if there are different budgets with access to the same fridge. Do we have it locked and hand keys out to appropriate people? Swipe cards? RFID-tagged post-docs? Now there’s an idea.

Non-random iTunes selection: ‘Summertime’ by Janis Joplin. A truly stunning arrangement.
Mood: Nectarine.

Actually, I think the standard of sales reps has improved in the 15 or so years I’ve been in this business. It’s been a while since I’ve been able to divert to the coffee room to avoid meeting a rep because I’ve smelt the aftershave lingering in the corridor.

It used to be really bad using a lift (’elevator’) after a rep had been in it, and heaven help you if you had to share one. . . friends of mine have asphyxiated because of the fumes. I used to work for a biotech company and dreaded on-site visits: Actually it wasn’t so much the visits, as I got to meet interesting people, but the travelling to and from the site in the company of sales & marketing (there’s a reason it’s ‘S&M’. . .) personnel.

I don’t know whether these people had questionable personal hygiene (hence the need for masses of aftershave/perfume) or they never turned on the aircon in their cars, but it was a crime against humanity. As I say, things do seem to have improved - none of the reps I’ve met in more recent times pong particularly badly. Maybe the companies make them shower daily now.

(Yes, I’ve been on holiday. Back to work on Monday - I might have something ’substantive’ to say then).

One of our favorite reps stopped by yesterday (where have you been Rob?) and other than a little chit chat, Rob handed me a flyer on “A Revolution in Resolution”. Basically it was hawking the benifits of a new acrylamide solution that is supposed to give you the resolution of a gradient gel without the pouring nightmares (not to mention “publication quality gels the first time…everytime!”). Unfortunatly we told Rob that we buy pre-cast gradient gels. Now this could lead to the whole topic posted yesterday about lab waste, but instead I will simply pass a conversation I had with hbogerd when I first started in the lab. We had done some cloning (we is misleading as I was a fresh rotation student), and needed to test for protein expression. I came into the lab in the morning and inocently asked when we were going to start pouring our gel (Westerns took two days in the old lab). “You’re living in the stone age” hbogey said with a grin, and then he led me to the cold room and the box of precast Tris-HCL gels.

The discard mentality is here to stay. Perhaps we can find better ways of recycling all this plastic we toss in biohazard bags. Kind of sounds like the discussions that took place as comunities moved toward recycling programs for household and consumer waste.

Below is our featured Perspective for this week, which I decided to cross-post in this blog because of its relevence. I thought that it would especially resonate with our Resident Bloggers. The Perspective is by Shirin Kalyan, Ph.D.

As a researcher in the burgeoning life sciences arena, I certainly appreciate the plug and play automation of the lab environment. However, I have not lost perspective on the goal of my work. A growing number of life science researchers have come to realize that our work for the improvement of health and our drive to understand the biological processes in health and disease is incompatible with the amount of waste we produce in the process. The desire to be a part of the solution and not the cause of the problem of biological “disorders” is a stark contradiction to the immense amount of unnecessary waste generated from daily lab operations.

It would be hoped that researchers with foresight strive to ensure that product stewardship and environmental conscientiousness is part of the business mindset of companies dealt with (although such a perspective would be considered optimistically-delusional, if not simply naive). However, there are some companies serving the life science community that have been fairly attentive in this matter and have even offered small token grants to academic institutions for the implementation of ideas to improve upon the sustainable research activities of users of their products, as well as making efforts not to use excessive material when shipping items. I am sure many researchers can identify with the ridiculous amount of packaging often sent with shipments of reagents and lab accessories. I have often been the recipient of boxes that could house mini-stereo systems only to find myself digging through an endless sea of Styrofoam packing chips to finally come upon a 1 ml vial of some research reagent. Such thoughtlessness in a field requiring some thinking and innovation does little to reflect the efficiency or competency of the company indulging in such garbage.

Communicating this interest to service providers would be a good start for ensuing change. I’ve discussed some of these concerns with local reagent suppliers and many have been more than willing to initiate programs (that would likely garner consumer loyalty) involving picking up packaging material from previous shipments and reusing them. Other companies are moving away from non-biodegradable materials as packing material. The environmentally sustainable researcher also is likely to be one who will reap financial benefits from saving money as well as trees – and there are resources available to help you be both conscientious and thrifty – such as online lab equipment exchange sites that have saved us a nice sum on shakers, evaporators and such necessities on the “eBay” for scientists. The turn over rate for technology is high in this field and the cost is often astronomical for our various gadgets – so it would only be fair, if not obvious, to have some kind of product stewardship program in place to ensure that the old centrifuge you discarded doesn’t end up at your local dump – where a scavenger may report it as a tool for the production of nuclear warfare that is traced back to you. One never knows, stranger things have been known to happen… and karmas have a tendency to run over dogmas.

The regulatory red-tape is also unburdened from the shoulders of investigators who take the initiative to use methods that bypass the usage of radioactive, carcinogenic, or otherwise noxious stuff for their assays. There are a number of more sensitive, cost effective, and less hazardous developments and tools of the trade (such as new fluorescence based proliferation assays that have replaced the incorporation of radioactive thymidine) that have replaced old standards (it is difficult to accept, but, indeed, everything old is not gold) – all it takes is a little innovativeness, a touch of awareness, and douse of initiative to plan laboratory activities in sync with the regenerative states of the ecosystem.

Our consumer driven shift to “disposables” is, frankly, a concept worthy of disposal itself. Unless there is a plan to recycle or reuse all these glorious “disposables” - it is neither an innovative nor a sustainable paradigm to adopt for the future of life science research. Considering the rapid growth of this field, those serving the research community would be wise to consider the consequence of the massive consumption and proportionally massive waste that is likely to ensue with such short-sighted development; and, if the ethical root of the argument of environmental sustainability is not enough to result in a change of heart, perhaps taking note of the history of companies that have fallen under the distrustful eye of public scrutiny and bad publicity would make industry providers take note. Biotechnology is an area that the public has yet to embrace whole-heartedly without suspicion of ulterior motives or being a field driven for profit and not for public good – especially after a few wrong turns with GM crops and the environmental impact of research and development. In addition to the general inert waste generated by disposable lab equipment in the form of various non-biodegradable plastics, the fact that the reagents used in research activity can be toxic when accumulated in the environment should not be ignored.

Some involved with the new exciting frontiers of nanotechnology have wisely taken notice from the get-go. Many embarking on being successful contributors in this young field have taken note of criticisms regarding the potential impact of their research and have paid due heed to public opinion and concerns about the unknown hazards of nanotechnology waste. As was eloquently expressed in a recent article published in The Scientist written by Vicki L. Colvin(1) , who is a professor of chemistry and chemical engineering at Rice University and director of the NSF-funded Center for Biological and Environmental Nanotechnology, stated “…ignoring reasonable fears and concerns about emerging technologies can halt or even derail technology’s progress. Industry now appreciates the costs of neglecting risks posed by new chemicals, materials, or devices…Safety and sustainability are no longer problems that concern only end-users well after the field is commercialized. Instead, they are flexible parameters in a new, and I think wiser, technology-design process.” Those lagging behind in this conceptualization, best take note lest they be left behind in the compost bin.

Reference Cited:
1) V.L. Colvin, “Research Vision: Sustainability for Nanotechnology”, The Scientist 18(16):26 Aug. 30, 2004.

Ever have an urge to paint your pellet? Me neither. Pellet paint from www.emdbiosciences.com is one of those products that I rarely use but every few months when I am precipitating a critical sample (forr example some mRNA isolated from some rare monkey tissue that I really don’t want to lose because the boss would kill me and I’d have to kill another monkey) I get it out of the freezer. Okay, I don’t really kill the monkey myself but I do request the tissue and I always wonder how much my monkey meat order influences the decision to execute little Bonzo.
Back to the details: Add a microliter of pellet paint along with the usual salt and 2 volumes of ethanol, freeze and spin. You’ll see a tight dark pink pellet which is the paint and your DNA or RNA. The pellet paint has never interefered with a downstream application and that includes RT, cDNA synthesis and cloning. I can’t remember how much it costs and one tube seems to last a lifetime.

One of the first molecular biology techniques I learned was the classic “alkaline lysis” miniprep. It is a very complicated procedure with three solutions involved which must be added in the following order-Solution I, followed by Solution II and then yes, Solution III. Got that? It goes I, II, III! Some genius at Qiagen http://www1.qiagen.com/plasmid/has devised a new and improved system which I think Qiagen should market as “Maxipreps for the Totally Incompetent” instead of “LyseBlue”. From the advertisement I saw, if you add your solutions in the correct order you get a COLOR CHANGE! I guess that is a real advancement for a num-nutz who can’t remember I, II, III. “Ah, crap my Qiagen did not turn color-it is still blue. I wonder if I added the solutions in the wrong order? I’ll just toss it and mess it up again tomorrow” Solution II lyses the cells (obvious visual change-clear and gooey) and solution III brings about the proverbial vomit chunks caused by the neutralization which lets you know you added I, II, III in the correct order. What about someone who is incompetent and colorblind? I guess they are out of luck, they’ll never know their Qiagen Maxiprep never was blue in the first place!

Book: “The Evil B.B. Chow” a collection of short stories by Steve Almond
Music: The Best of Phil Ochs
SAB Longevity Levels: High
SAB Amusement Level: High
Science Mood O’ Meter: fluctuating wildly
Experiment holding up a manuscript: Sucrose gradient

Just to add to the glowing operon review, we typically get very high yields from even their smallest scale preps, so we almost never need to order anything larger. We have only had a couple problems with oligos ordered (one failed quality control and took a couple extra days), the vast majority show up on time without hassle early in the day. I think the Fedex shipping helps with this.

over all, two thumbs up!

For those of you who order oligos routinely, I highly recommend using Operon. Operon offers pre-paid CD’s which makes ordering extremely easy. They set an online account up for you which shows the credit balance you have with them. Any time anyone in the lab orders oligos, they log into our account, type in the sequence and the price of the oligos is automatically deducted from our credit.

I then get an email telling me what was ordered and by who (when we order oligos everyone must name them beginning with their initials). Additionally, I receive an email when the order ships and then one from FedE x as well. If there are any problems they also email to update on shipping. Usually, you get your oligos in 2 days.

If you order as many oligos as or lab does, Operon makes ordering as painless as possible! I would definately give them a try.