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These are the real things that give nightmares to post-docs and graduate students.

One thing that you don’t learn, until you either do a research project in a lab or you start graduate school, is that science isn’t really the straightforward cut and dry: we do this step, then we do this step; sort of field that you might imagine. You come to graduate school all bright-eyed and amazed, with the scientific method burned into your brain, only to find out that it’s usually the little things that no one told you about that make the difference between productive experiments and a waste of time.

Learning it from your friends
Just like sex education in a religious household, sometimes you have to learn lab technique on the street. One example that stands out was the year I took Quantitative Analytical Chemistry Lab. Actually, I took it twice. I enjoyed the class the first time, but had to drop in the middle of the quarter. The second time, I had been working part-time, for a few months, as a lab technician and had learned a little more in the interim. For example, the second time, I knew about stir plates and stir bars. None of the chemistry TA’s had ever said anything to the students about using stir plates, but once I realized what they did and learned that you could check them out from the chemistry stock room, my data got much better, and my friends and I all got A’s.

Systematic errors
Sometimes there are obvious things that people do wrong. Like the guy in a friend’s lab who always grew his E. coli cultures for days on end before trying to isolate DNA. I think he eventually switched to a department where he could work with non-living biochemicals.

Then there are things that everyone thinks that they must know because they seem so obvious. Many people use micropipettors wrong. For some reason, they miss the part where you only press the plunger down to the first place it stops, then let go, then press the plunger to the second place, where it stops, to expel the last bit of liquid. Oh no. They enthusiastically push the plunger all the way down and suck up about 25% more liquid. When I was teaching in the biotech lab, I learned how to catch these problems. I would give students 1.5 ml microcentrifuge tube, with volume markings, and have them pipette 750 microliters of water. They had to get the correct volume to pass the course. Some of our students , who had been working in research labs, were shocked when they found out that they had been misusing pipettors on a regular basis.

I also learned that it was possible to complete 4 years of an MD/PhD program without finding out that a solution of 30% sucrose, with 30 grams of sucrose, should have a final volume of 100 mls. Luckily, molecular biology is pretty forgiving and now there are kits for many procedures, so those people can get by without knowing how to make solutions correctly.

The dangers of lazy decisions
Later, I realized that there were differences in technique that couldn’t be easily sleuthed. One case was a post-doc in my lab (where I was a graduate student) who was trying to do RNAse protection assays. It was a complicated assay. First, you had to make radioactive RNA by mixing T7 RNA polymerase, a plasmid, and some radioactive ribonucleotide triphosphates. Then, we precipitated the RNA and counted the radioactivity to determine how much we’d made. Later, we would add the hot RNA to the DNA we were studying, add some RNAse to digest unprotected RNA, and run the results on a gel, alongside with fragments of hot DNA, to locate the positions of gaps or nicks in the DNA.

With all those steps, there are many places where this sort of thing can go wrong. And this guy kept a pretty useless notebook, so his notes were no help. He tried and tried. Nothing worked. I think four or five of us were doing the assay, and getting it to work, but not him.

One day, he came into my room (our lab had several small rooms) to precipitate his RNA and I happened to watch what he did. He set everything up properly, then grabbed the water bottle off of my bench to do the wash step. “What are you doing?!!” I yelled.

I’m washing the RNA with ethanol

“No! You’re not! You’re washing it with distilled water!”

Well, someone had written a faded, scribbly “Etoh” on the other side of this old wash bottle long ago, but it wasn’t me, and I certainly didn’t feel obligated to obey someone else’s ancient scribble. It was my bottle. I knew it contained distilled water. The guy who was stealing items off of my bench did not.

What do we do about this? Look for an answer to appear in an upcoming post.

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Comments

  1. #1 Pharma Bawd
    November 1, 2006

    Like the guy in a friend’s lab who always grew his E. coli cultures for days on end before trying to isolate DNA.”

    Probably a friend of the chemist who figured he could get twice as much DNA if he grew his E. coli at 74C.

  2. #2 Julie Stahlhut
    November 1, 2006

    My husband and I once had a pharmacist at a major chain drugstore tell us that he’d have no idea how to make a 0.4% solution of a drug that was normally shipped at 2% concentration. After we scraped our jaws off the floor, we located a real compounding pharmacy within driving distance that could fill my prescription.

  3. #3 Beth
    November 1, 2006

    What do we do about this?

    Smaller lab sections, better pre-lab discussion for undergrads, and quit stealing off of other people’s lab benches!

  4. #4 PhysioProf
    November 1, 2006

    “Luckily, molecular biology is pretty forgiving and now there are kits for many procedures, so those people can get by without knowing how to make solutions correctly.”

    In my opinion, kits have a very dark side, in that they allow–and sometimes even encourage–ignorance about what you are actually doing. This makes it very difficult to troubleshoot when things don’t work. I allow trainees in my lab to use kits, but I will upbraid them if they cannot explain the principles by which the kits work.

  5. #5 Sandra Porter
    November 1, 2006

    Pharma Bawd – oh! that is great! I can picture that sooo easily. In fact, this guy DID switch from microbiology to the chem dept.

    Julie – The funny thing about the guy who was having trouble making 30% sucrose was that he didn’t believe me when I told him how to do it properly. But he came back with his tail between his legs and apologized after he got the answer from another post-doc in a different lab.

    Beth – Oh, I have lots of ideas on this subject, but it will take time to get them all down in writing.

    PhysioProf:

    In my opinion, kits have a very dark side, in that they allow–and sometimes even encourage–ignorance about what you are actually doing.

    I agree. We never used kits when I taught the biotech lab. The students made their own media, solutions, and buffers. Life is complicated enough without having to determine the purpose or identity of mystery reagents, like solution A and solution B.

  6. #6 Brian
    November 1, 2006

    This guy was screwing around with radioactive RNA and a wash bottle? Never mind that it was your wash bottle – there’s just so many things wrong with what he was doing to begin with. He was just asking for P32 to splash onto him or introduce RNase into his preps.

  7. #7 Sandra Porter
    November 1, 2006

    This was quite awhile ago, but precipitating radioactively-labeled probes onto a filter for counting, was a pretty standard technique. You would set up the filter apparatus near the sink, hook a hose up to a glass arm on the flask and turn water on to apply suction to the filter. Then you would put a small amount of your probe on the filter and use an ethanol wash to remove unincorporated nucleotides.

    After all of that, you would put the filter into a scintillation vial and count the amount radioactivity that had been incorporated into the probe.

    The post-doc’s problem is that RNA is soluble in water. So, it always looked like he had screwed up because he didn’t see any labeled material on the filter.

  8. #8 jake
    May 18, 2010

    hi im only 13 i was looking up home work stuff and your story sound well like a story like a book i wish you wrote more i enjoyed reading :)

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