Life Science Tools of the Trade

There have been a couple of posts about Operon oligos in this blog.

Quick summary: short oligos (~30 nt) they do a good job with quick delivery (usually 2 dya delivered). However I recently made two clones using 79 mers. I made the clones but found that 3 of 6 clones I sequenced had one or two mutations introduced by the Operon oligos. Another odd thing is you are forced to buy a larger scale. Okay, that would make sense if you need to scale up to get enough full length product. I received tubes with approximately 4 milligrams of oligo! After I use one mgm I can repeat the cloning only 3999 more times if it doesn’t work the first time!

I’m not sure who provides the best quality long oligos but I’m pretty sure it isn’t Operon. Since the mutations were substitutions paying for HPLC purification would have been a waste of time. I’ll look into Sigma/Genosys and Dharmacon and let you know in a future post if they provide a better quality product.

SAB Mood O’ Meter: Nexins returns (a good week)
Science Mood O’ Meter: Does it get any worse than this week? #!^%, I hate science (and just wait until the paper gets rejected).
Music: CD I purchased but I’m not listening to Big Star’s reunion disc entitled “In Space”
Music: CD I purchased and I’m enjoying, Laura Cantrell’s “Humming by the flowered vine”
Novel: “The Greatest Man in Cedar Hole” by Stephanie Doyon

I recently tried GenCarrier1 and have to say I am less than impressed. In parallel I tested Fugene and the much cheaper Gen Carrier1 transfection reagent. Verdict: I just came back from the microscope and ~18 hours post-transfection (3T3 cells) I see tons of green cells (Fugene) and none, not one with GenCarrier1. Yes it it 1/2 the price but it doesn’t work even half as well! I’m often willing to try a new reagent. However, it damn well better work the first time. I already have the system going and I’m not about to spend my time optimizing a protocol that may or may not ever work to “potentially” save a few dollars.

Additional comment: I called the company (Epoch Biolabs, Inc.) and left a phone message. Never heard from them thus I have renamed their product GenCarrion1 in honor of their nonexistent customer service.

I helped a guy in our lab spec a new iMac at the beginning of August, and he put the order in a few days after I went on holiday.

It’s only just arrived.

I’ve ordered a top of the range G5 and 23-inch flat screen so that I can solve the solution structure of the protein I wrote about last week. Order went in on Monday. Any bets as to when it’ll turn up? And I’m out of here at the end of the year. ARGH.

Apple - great products, lousy company. I guess it’s part of the mystique: You’ve got to really, really want one before they’ll ship. They’re probably running background checks on me as I type - “Is this person good enough for a Mac?”.

Two fairly friendly sales reps stopped by the lab the other day asking about our lab water. I told them how much we pay for our service contract wich includes maintenance and the cartridge/filters. They quoted me a price that was a little lower than what we pay now. It slowly dawned on me that they said they would be competitive for “consumables” when they walked in the door.
They neglected to tell me until the very very end of the conversation that we would have to buy their $4400 system to save $200 per year. What a couple of used car salesmen dirtball sales reps. Why do they need to send them around in pairs? Perhaps if they realize that if they waste the time of the wrong person someone might just kick their @$$.

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?

Science doesn’t stop even when you’re trying to wrap things up for vacation. I’ve been finishing up some immunofluourescence photos so that I can sit back and relax on the beach next week. After doing the experiments and then trying to take the photos the hard, I asked the lab next door if I could use their microscope setup. I have to tell…Man was I impressed.
The system has an olympus camera for the photos but that wasn’t what impressed me the most. What I found truly amazing compared to our system was the imaging software package. Taking the photos is a breeze. The image comes up real time on the computer monitor and you adjust the focus, exposure time and black/white balance and click. The image is then imported into the imaging software, DP Manager.

Now there really isn’t much to manipulate after you take the photos but the nice thing that this software is that it does overlaying. Once you click the option to overlay, you simply toggle back and forth between the photos you want superimposed. You can superimpose your FITC stained photo right on top of your TRITC and your DAPI stain or any combiniation there of. 99% of the time the overlay is perfect but if you should need to move the photo left or right you just put the coordinates in to do that. More importantly, you can increase or decrease the intensity of one photo to match the intensity of the other.
I admit before using this software I was impressed by the photos you see where the nuclear is dapi stained and the cytoplasm is Tritc stained and then there is a third stain. I figured someone spent a lot of time and effort to achieve that photo and they might have. With this imaging system however, almost anyone can do it.

Sorry for the lack of blogeration. I do have a good excuse. Yep, I’m trying to figure out how to podcast this blog.

I feel liberated having finally truly given Qiagen the boot. The irony is that the new sales rep actually cared about us as customers. Of course, no one in the research and fuzz developmentat Qiacrap is going to call us and say “Hey, we’ve been doing this for years and we have no clue what that crap is”. Now they may well call and say “We have a new and improved Plasmid Purification kit and do you want to try it?”. NO I DO NOT! Just like when McDonald’s basically confessed that their “McNuggets” were ground up chicken gonads when they came out with their new and improved “Select Chicken Strips” I take a new kit as a confession that Qiagen has known for years that their product had a problem.

I am not a big fan of lipid based transfection kits. I recently gave Fugene a try when I had a cell line that was barely transefectable with the standard calcium phosphate procedure. To my very pleasant surprise 90% of the cells transfected with a GFP reporter plasmid were green 48 hours post-tansfection. Note: No need to remove antibiotics or change media is a real plus . Viability appeared to be over 95%. I also tried Lipofectamine 2000. 48 hours post transfection the 5% of cells that weren’t dead or dying were transfected. Is that considered 100% transfection efficiency? Blah.
Conclusion: I like Fugene (another Roche).

Music: Richard Thompson “Front Parlor Ballads”
Novel: Alan Zweibel “The Other Shulman”
Movie : “The Hitchiker’s Guide To The Galaxy”
Book on tape: “The Hitchiker’s Guide To The Galaxy”
SAB Mood O’ Meter: calm, quiet
Science Mood O’ Meter: to be determined, still waiting for reviews
Today’s Pet Peeve: a really really big fat bubble in the middle of my Northern blot

Our Institute changed tip suppliers over the summer. As you might imagine, with 400 scientists it’s not a small contract. The lucky people (STARLAB) who won the contract to supply our Stores don’t distribute the best tips on the planet, but I don’t really care since The Powers That Be wouldn’t stock the low-retention plastic ones I’d like in a month of Sundays. If I want them I’ll order them myself, as it would be a bit expensive to have them as our routine supply.

While the trials were ongoing (you think we don’t trial them before Admin make a cost-based decision anyway? Shame on you), the STARLAB reps picked up on the fact that many of the people who tried their tips were complaining that they kept falling off their pipettes. So when they landed the deal they came in and said right, we’ll calibrate and service all your pipettes (Gilsons and others) and replace the barrels if necessary. For free.

That was nice, and they steamed through hundreds of the things in jig time. I don’t know how thorough the service was, but my pipettes got new ‘O’ rings and seals, and the calibration certificate is pretty.

Of course, the day I got my Gilsons back from the STARLAB guys I got (yet another piece of) junk mail from Anachem, offering to service my Gilsons at a reduced price. Ha. Thing is, the service from Anachem is hardly cost-effective; after three years it’s cheaper to dump the old ones and get replacements, rather than having them serviced. Someone needs to wake up to this.
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It must be “Bash Qiagen Week”. Makes a change from “Bash Microsoft Century” I guess.

Chap in the lab has a (free, I assume. There’s no other reason to wear it) T-shirt from Qiagen that boasts the motto “Innovation in Bioseparation”. And I can only assume it’s not talking about the big Q because they haven’t innovated in fifteen years or more.

Let’s see, when I was working at a DNA separation technology company I revamped their kit product line. One of the improvements I made to the miniprep kit we sold was to include ‘Eppendorf’ tubes with extra long linkers between the tube and the lid. This was so that users could cap the tube with the filter bucket in place, obviating the need to cut the lid off when eluting the DNA at the end of the prep: maybe not a big change but it addressed something that annoyed the goolies out of me when performing Q minipreps, not least because I tend to write on the lids of eppies rather than the sides.
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