Things that go wrong in the lab

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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