Life Science Tools of the Trade

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!

Never heard of this company before but somehow their catalog found it’s way onto my desk.  You know me.  Usually, I’d just recycle the catalog and forget about it.  We don’t buy anything from this company and I had no clue what they sold.  The thing is:  there was something different about the catalog.  The cover has a funky design and says envision proteomics.  Not overly helpful, that encompasses a wide range of products, but still, I found myself thumbing through the catalog. 

As I’ve learned, Abgent is an international company with its US distributor based in San Diego.  The company sells antibodies, specifically, antibodies to post translational modifications.  You know the antibodies.  Those to sumoylation, methylation, phosphorylation, etc.  Additionally, Abgent has custom services for peptide synthesis and antibody production. 

So, now that you know all that.  The thing that really struck me about the company was the catalog.  For each antibody in the catalog there is a western and/or an immunohistochemistry photo.  You know the saying, a picture is worth a thousand words.  Some of the antibodies are very clean, one crisp band.  For some, you see background bands.  The thing is:  the figure/data is right there.  You know what you’re getting and you know if it’s got background.  No surprises. 

Kudos to the marketing individual that opted for including all that data. 

I’m sitting here, trying to work out how I can mutate leucine into aspartate and simultaneously introduce a restriction site so that I can check if the mutagenesis worked with a simple digest rather than having to wait for sequencing. Let’s see . . . TTA to GAT, and there’s ATC before that - TCGA is palindromic, plus A and T . . . ClaI!

Page 214 of the NEB catalogue, I salute you.

I’ve known labs that don’t even use New England Biolabs (NEB) enzymes keep a copy of their catalogue. On the off chance that you’ve been living on Mars for the last decade or more and don’t know what I’m talking about (say ‘Hi’ to the rovers for me, and what did you do with Beagle 2?), the NEB catalogue is an indispensable reference. It is little exaggeration to claim that I use it every day when I’m doing molecular biology.

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In a new lab it’s important to find out where the catalogues are. Yes, many companies have online catalogues, but you can’t beat holding one in your hand, thumbing your way through it, taking note of the coffee stains and the thumb prints (something blue on the ‘C’s) and the little scribbled aides de memoire. These are often totally illegible with time yet provide a link to times and postdocs past and future; a communion with the very essence of Science.

It’s also useful when someone else has already worked out for you exactly which of the thirty-one grades of bovine serum albumin in the Sigma catalogue is appropriate for blocking your plate. You could probably ask your lab manager but she always got the postdoc - yes, your predecessor, who left the country in rather a hurry and took all his notes with him - to order it, and it’s no good asking the boss because, let’s be frank here, bosses inhabit a world that’s consistently pi out of phase with Real Lab Life (TM).

Of course, if you’re setting up a lab (and you haven’t managed to steal catalogues from down the corridor) or you’ve finally got around to replacing the Sigma 1997 edition with something a little less dog-eared you’re going to have to do this from scratch yourself. If you annotate the catalogue, half an hour now spent making sure that this is the correct version of DNase means you won’t have to do it again. It’s the same principle that is behind gene annotation. If someone else has labelled it already you don’t have to do the experiment. Score!

I used to have a pad of those little sticky coloured strips, ‘Post-it’ notes for books, that I’d use to flag pages (and especially the New England Biolab one - more on that next time) or to scribble little messages (like ‘100 g phnl 45s3′. Hmm. My pen smudges on these things) so I - or the grad student I’d just asked - knew what to look for. Now my catalogues all have little yellow mohicans, I can’t read what they say, and I can’t start again because some inconsiderate so-and-so has swiped my pad of sticky strips. Excuse me, I’m just off to have a word with the lab manager.