Life Science Tools of the Trade

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.

Okay.  I didn’t think about it.  I guess nobody did.  Since when do you have to specify inside delivery on an order?
The other week, I ordered a fair bit of pipet tips.  Turns out, the order turned out to be a pallet worth of tips.  The company shipped them freight from California.  Probably cheaper shipping costs for them and it doesn’t matter to us how they’re shipped.  What does matter to us was that they were delivered to the lab.  They were dumped on the loading dock.
The truck driver actually came up to the fourth floor to inform us that he didn’t have orders to make an inside delivery.  All he had to do was leave them at the loading dock.  Okay.  Not thrilled about it but not a big deal.  We could go down and get them.  The irritating part of all this was when he offered to deliver them upstairs.  With the use of our cart and for an additional fee, he’d bring the order upstairs.   WHAT?!?

I still can’t figure it out.  Was this a way for him to make a little extra money?
Needless to say.  We didn’t pay him to bring the tips upstairs.  Three of us went down with a cart, pulled the pallet apart and *delivered* the tips the lab.  One cart, one trip, no extra delivery cost.

Nope, you’re right.  I’m not a believer in having *special* enzymes for a quick restriction digest.  What caught my eye about this particular group of enzymes is the buffer they cut in.
Fermentas has come out with a group of fast digest enzymes that all cut in the SAME buffer.  EcoRI, MfeI, HindIII, BglII, BamHI, SalI, XhoI… all enzymes I routinely use.   One buffer for all of them.  You no longer have to consult buffer tables or charts!  Simply set up your reaction and digest away.

Disclaimer:  My understanding is that they are special enzymes NOT a special buffer.  You can’t use your other enzymes in this buffer and assume they will work.

that decided to sell FuGENE 6 in these bottles? 

I was just in the hood mixing my 5ul of Fugene with Opti-Mem when what do I do?  I tip the bottle of Fugene over and spill half the contents out.  Admittedly, I’m a klutz but the bottle doesn’t fit into any of the racks we have in the lab.  In case you don’t know, it’s a tiny glass bottle with a screw top.   

Whose idea were these bottles anyway?  I really like Roche as a company but they dropped the ball when they started using these bottles!!

Wotcha folks, long time no see.

I’m going to cheat here, and link to a piece I’ve just written elsewhere, because it fits well under Tools of the Trade.

The word alone chills the hearts of experienced cell biologists. And when, a couple of months back, someone upstairs was getting strange results from their cell-based assays, the Boss came to me and asked,
‘What do you know about mycoplasma?’. The icy black hand of dread gripped me and I spent the rest of the day trying to find a supplier for a mycoplasma detection kit in Australia.

Please forgive me for this blatant self-plagiarism.

Everytime I walked down the hallway to the lab, I kept seeing the ad for the new HaloTag system.  Well, finally, it got the better of me.  At the last vendor show, I spoke with the company sales rep about it.  She was helpful and provided me with some literature and one of the plasmids for cloning.  Thanks! 

If you haven’t heard of this system, the simplistic explanation is this:  you make a fusion protein of your gene of interest to an engineered derivative of the hydrolase gene.   Now that your protein is tagged, you express it in mammalian cells and then purchase differently labelled ligands to visualize it. 

What I’m not sure of… what’s the advantage is to this system?

The ligand covalently binds to the Halotag and comes in many flavors.  You’ve got your Biotin ligand, your coumarin ligand, your FITC ligand… Are you getting the picture?  You need to buy a different ligand depending on your experiment.  Presumably, how they plan on making money.

I was most interested in the immunofluorescence technology.  If it was going to save me time and frustration, I was all about changing over.  However, after reading a bit more, I didn’t see an advantage over traditional IF.  After fixing the cells, you bind the FITC ligand to your fusion protein and visual it with the scope.  How is that different than fixing cells and incubating with a FITC/TRITC conjugated antibody? 

The bottom line.  Either I’m missing the point of this technology or putting any tag on your protein will get you to the same spot.  I don’t see what a hydrolase fusion gets you over a myc or flag or HA tag. 

If anyone knows of a true advantage to this system or has actually used it, please let me know.  Until then, I’ll keep using my HA fusions for my experiments.

Maybe someone can explain this Hybridization buffer to me. I buy it for the post docs in the lab but I really don’t understand the advantage of it. My understanding is that it allows for quicker hyb times than traditional buffers.

Sounds great BUT what perplexes me is that they have to incubate the ExpressHyb for hours for to get it to resuspend. How is that faster? By the time the buffer is back in solution they could have already hybridized the blot with the old fashion cheap buffer. That’s the other thing, this stuff is expensive. $500 for 1 litre!

Right now we have the money so they’ll keep getting the ExpressHyb but with NIH slashes….who knows.

The lab supply of FBS was dwindling and we decided it was time to stock up again. I called the company we had previously used to get a quote and found the price went up over $80 for 500ml!! This of course resulted in further quotes and looking into other companies and what their FBS was.

I can fully admit after all that I’m even more confused and amazed at the different types of FBS that are available. Low, mid-grade, and high endotoxin, heat inactivated or not, defined or not, US origin or not….the list goes on and on and the prices vary considerably.

My confusion led me to the cell culture facility here and a VERY important lesson. Most labs purchase FBS based on the company and not the serum itself. I admit, I didn’t end up getting the cheapest stuff. The old saying, you get what you pay for and all that. That being said I didn’t go for the most expensive either. I opted for the middle of the road serum and found out that’s what the facility uses for most of their cell lines.

I guess the point of all of this rambling is….if you are trying to save some money, make sure you’re not wasting it on FBS that you don’t need.

What reminded me of the farce I went through in my previous job was a mailing from Flowgen, who are distributing (in the UK, at least) Gentra’s VERSAGENE™ range of DNA purification kits - genomic DNA that is, from blood.

Superficially it looks like a method that was developed at the company I worked for about 7 years ago. The methodology was based on a particular filter paper from a well-known filter paper manufacturer that had special properties. The manufacturer had a line of forensic papers that it supplied to police forces around the world, and we were particularly interested in DNA profiling (we had a state of the art ABI machine and could do SNP analysis, fingerprinting, the works).

So we had this method that involved 1 ml whole blood, two solutions, a column, a heating block and 10 minutes. Whole blood was applied to the column (which was basically a holder for this filter paper), sucked through under vacuum, lysis/binding solution added, suck, wash with same, suck, add water/TE and elute with heat. This last stage I seem to remember did not require negative pressure; the eluate just dripped through. And all this happened in under ten minutes. Yeah, the DNA was shagged - single stranded and 20 kB max - but it gave beautiful PCR data. Probably because it was single stranded.

It was a world-beater, but marketing were not happy. Oh no - my brief was to scale this up to handle 10 ml whole blood, using exactly the same technology (and also apply the same technology to bacterial cultures for plasmids, yeast, faeces . . . - I kid you not. That’s the kind of mentality I had to deal with), handling twenty four (24! That’s a whole unit of blood!) samples simultaneously. I played around with it for a week and announced that it could not be done. It wasn’t possible to elute the DNA from the filter in columns that could handle 10 ml blood without cooking it completely. It could be done if the protocol could be extended to an hour (the time it took to filter the solutions was much longer than for the 1 ml prep - this is obvious if you know anything about vacuum pressures and cross-sectional area), but even then yields were pretty poor. Nope, marketing wanted 10 minutes and no black pudding.

I wasted six months of my life on the project before landing a job I saw advertised in Nature and was replaced by two people who still hadn’t got the 10 ml scale prep working two years later.

But the point is the bloody management insisted that we stuck with that particular filter paper, despite me clamouring that we needed to try something else. I swear the CEO was on the take. Had we been able to find a more suitable matrix we’d have had a decent product, without an RBC lysis step and five solutions, five years before Gentra came out with this kit which - let’s be frank - is early 1990s technology at best. And this is symptomatic of the industry - there has been no real innovation in DNA extraction technology in ten, fifteen years. Yet Qiagen, Promega, the rest of them are slapping us with 800% markup on each and every prep they sell.

You’ll forgive me if I’m somewhat disillusioned.

Of course, it’s not just the science that requires reliable suppliers of quality equipment and consumables. The scientists, too, need victualization. I once read on Usenet that the four food groups are sugar, salt, fat, and chocolate. Caffeine and alcohol, however, are among the essential vitamins (Steve VanDevender).

Of these nutrients, scientists generally can not do without at least two, and usually three, of them on a weekly or more frequent basis. Free pizzas at trade shows and grad student soirees satisfy the fat and salt requirements, but this phenomenon is not widespread outwith the continental US. Caffeine, on the other paw, is consumed widely with much ceremony and gratefulness the world over on an incident timescale of minutes to hours. Alcohol is frequently used as a broad-spectrum analgesic and muscle relaxant, and when diluted with water is also very useful for cleaning and sterilizing benches. On average, we are looking at a timescale of hours to days here. Ultimately, the scientific world runs on various incarnations of the humble cocoa bean, and that my lab mangler does not like it is further proof of her continuing contribution to the oxygen deficit.
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After three weeks’ sweat I finally got my protein sample, labelled for NMR, 99% pure at 10 mM in the fridge. It’s been difficult, the student couldn’t get the thing to express in minimal media so I was brought in to try, and after an epic battle which involved suspect TEV protease, chloramphenicol-S-Transferase and finally doing things the old-fashioned way, I got there. It goes on the magnet on Monday.

But what I came here to talk about is the supplier of the 13C-glucose and 15N-ammonium chloride we use to label the protein. Spectra Stable Isotopes are the guys, and they offer a not unreasonable price tag with their reagents. 13C-glucose, for example, is ~US$140 per gramme, so my experiment came in at about 600 quid, which is a bargain really (especially when you consider that I ended up with 150 mg protein at the end. What am I going to do with it all?). How do they do it?

I’ve often wondered about making these labelled compounds (and they do amino acids and suchlike, too). Organic synthesis? Euch.
Well, apparently they can synthesize complex molecules, but for most of the catalogue they let Algy do the work. Not Algy, algae. They feed isotope-enriched salts and carbon dioxide to algae, shine the sun on them and let the little blighters do all that boring intermediary metabolism stuff, then smash them apart and extract labelled goodies.

Clever, eh?

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.

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.

Remember the bad protein marker story?

I ordered some SuperScript II (not III) on the advice of a post on the Scienceboard. It arrived in timely fashion, but the tube of 0.1M DTT was empty. Not a major problem, but I’ve paid for it so I damn’ well want it. An email to the tame rep resulted in a vacation message, so I called the support line and got them to sort it out. I had to email the order number, etc., and then I got an email from someone else at Invitrogen asking for the same information, and duly replied.

On consecutive days later in the week I received a new SSII kit and a M-MLV RT buffer pack (which contains DTT). So I’ve ended up with two SSII kits for the price of complaining. I guess the rep must have chased the problem up when he got back, setting a second set of wheels in motion. I’m not complaining, mind.

Oh, and apropos nothing in particular - if you’re a publisher or a journal marketroid, it takes more than one bottle of wine and three small baskets of nibbles in the foyer to get 400 scientists interested in your flagging journal. What a surprise no one stayed to talk to you.

Now I’m not saying that our labmangler is technically deficient - she’s been in the business 12 years after all - but how is it that she had trouble with BamHI and I didn’t?

I cut my vector and insert last week, set up the ligation (using minimal amounts of ligase, natch) and got a nice number of colonies on the appropriate plates. Did some minipreps, and went to the freezer to get some Bam to check for my insert. Uh . . . this looks like a new pot of enzyme. Why is there a new pot of Bam? The ‘old’ one was nearly full, and I know we don’t go through that much.

Turns out that our labmangler had trouble with Bam - if it cut at all, she was left with nucleotides. So she threw it out and got a new pot.

I can understand that; after all, it’s cheaper to get new enzyme than spend a week trying to figure out what’s wrong. But that batch had been working fine for me! I used the new pot anyway - what choice did I have? - and exactly half my minipreps had the insert I was after. So the new enzyme works, too.

I asked her this morning if the new enzyme worked for her.

“Sort of.”

Not very encouraging. What’s the difference? It’s the same buffer. Hal will laugh here, but I routinely add spermidine and BSA to my digests. Always have done, ever since I was a pup of a grad student. It saves looking up which enzymes actually require supplementing, and it doesn’t hurt, so what the heck. I’m wondering if this is real evidence that it is actually making a difference. The alternative explanation is too horrible to contemplate.

We had a batch of dodgy protein marker from Invitrogen. Back in January, I think it was. I made a phone call to their support desk (”We’ve not had any complaints” “You have now”) and extracted a promise of some replacement marker. Months passed, and I nearly forgot about it, until I was introduced to the new rep (I’ve talked about him previously) and in a flash of quite uncharacteristic brilliance remembered to quiz him about the replacement marker.

Turns out (after I emailed him a reminder, grr) that they’d closed the ticket because they’d sent out a replacement that, naturally, I’d never received. Got on the blower to the tech service girl, who says they have not had any complaints about the replacement batch.

“But I never received it.”

“That batch is all gone now, but there were no complaints.”

“That’s not the point . . .” after going round in circles for a bit I persuaded her to send some more (maybe it was the bit about ripping someone’s head off and pissing down the hole that convinced her) which, much to my surprise, arrived three days later, with my name on the parcel.

Score. Of course, they had the last laugh by sending two customer satisfaction survey emails out to me. I filled in one of them - and to be honest I was quite pleased with the (eventual) outcome, but I have this mental image of two tubes of Benchmark Ladder languishing somewhere in a warehouse near Milton Keynes, with no one to love them or give them a home. Poor things.

Things I must remember to blog about this month: Invitrogen versus Qiagen, Our Crazy Ordering System Part II.
Mood: Green
RiTS: They Don’t Know by Kirsty MacColl

Note added in proof:
IVGN are doing really well for themselves, aren’t they? They’ve landed the first licence in ABI’s expanded licensing system, whatever the hell that is.

I’ve had a bad week.

My bike died, the frame is slightly bent, and being alloy I’m a bit leery about it being straightened. I hope the insurance assessor will agree with me. Then my external HW drive that I use for backups and iTunes at home made a *clunk* noise and won’t spin up. Nearly everything on there is backed up, except for 100 photos of the Royal Botanical Gardens at Kew and a dozen or so taken at the Loved One’s graduation. Oh, and the latest draft of a novel I’m working on. Gah. It would cost anything up to a grand to recover the data, which is worth it to me - if I could afford it. Bugger.

On the upside it’s a bright sunny day for my mum’s birthday today, but the bad news it’s already 31C (88F) in the lab and it’s not 9 o’clock yet. Aircon? What a great concept. Bah. I think I’ll move the computer to the cold room.

Anyway, beloved reader, you didn’t come here to hear me whine. We do have a products & suppliers piece of news this week. I think I’ve mentioned before that we have a rather good Stores in this building, who stock pretty much all of the day-to-day stuff we need. But in a somewhat radical move (at least for this institute) we - that is, our lab and a couple of other groups on this floor - are getting together with Invitrogen (the 500 lb gorilla, etc.) to implement a mini-stockroom in the cold room I just mentioned. The idea is that we’ll have a ready supply of gels and markers even when the rest of the building is scrabbling around because someone central hasn’t placed an order or paid an invoice (and both have happened, I assure you).
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This post has been sitting in my ‘out-tray’ for ages. I’ve got a rant about Qiagen brewing that will probably spill over into two episodes, I’ve got the second part of the Ordering System madness to get off my chest, and I have to go to the dental hygienist tomorrow morning. So, I’ll throw this one to you hungry beasts.

In a previous existence I used Boehringer Mannheim (who were swallowed by Roche) restriction enzymes and made my own buffers. Five different buffers (L, M, H, 1 and 2 I think), 50 ml stock of each, 1 ml aliquots for use. Everyone I worked with agreed that BM made the best enzymes, and places like Promega and NEB were rank upstarts with no QC to speak of. Of course, no one in their right mind would even have considered using Sigma enzymes.

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Some people here at the Institute for Medical Advancement have had problems with their Southern blotting. Apparently this isn’t down to the usual culprit (*cough*user error*cough*) but rather a bad batch of Amersham’s Hybond(TM) N+ membranes. This surprises me somewhat, Amersham having had a good reputation for quality (and corresponding expense, but you get what you pay for) in the past. I must admit, I was also a little surprised last year when General Electric bought Amersham, given the latter’s stranglehold on quality radiochemicals in this country and its commercial success since it was privatized in 1982 (to such an extent that it was able to swallow Pharmacia whole, without getting indigestion).
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