PCR happy ending


2014-01-17 18.04.26

Me taking a picture of the envelope taped to the front door before leaving Friday evening. Just in case.

When I wrote the previous post about my anxiety regarding the PCR reaction of the soil cultures, I did not explain the main reason for it: this was to be part of an actual laboratory class. Those of us who teach lab classes know the feeling of slight embarrassment when things go awry. You expect a reaction that does not happen or the microbes don’t look the way they should. Reasons abound: from human mistakes and carelessness to expired reagents, bad batches, or plain biology. Sometimes it can be made a teachable moment, but not always.

So last Thursday I went to the lab hours earlier and did a mock run, which was a good idea, because I made a number of mistakes, one being walking away after pressing the Start button. Turns out the machine wanted me to press an additional OK, then asked about 2 more settings, and finally made me confirm that I was ready to run. Luckily after half an hour I had started wondering why the machine was so quiet. Duh.

By the time the students rolled in I was feeling much better, and after pipetting the reagents of the first tubes, I asked for volunteers. There were several, and as they were enthusiastically pipetting the 1 ul volumes I started wondering if that was a good idea. None of those students had ever used a micropipette before.

But they were delighted and enthusiastic, something that all participants of the Small World Initiative have reported. Ownership of a project comes hand in hand with motivation, and once the tubes were in the machine and the students returned to their plates it was a joy to watch them discussing their soil isolates.

When I ran the electrophoresis the next morning, I was a bit disappointed but not surprised. One lane had a clear band, some had faint bands, and the rest were smeary. As I expressed to one of the students the day before: we want the first experiment to work, but not perfectly so we can improve it for the next time. A first flawless experiment is almost always a fluke, and we cannot learn from it.

So I ran the samples again, this time in the blessed quiet of the lab. Things looked much better. I called the company that would do the sequencing, and scheduled a pickup around 2 pm. Put the samples in an envelope, left them at the front desk, and went on with my things. At 5 pm, the envelope was still at the front desk. Called the company, and they told me the courier was on its way. “LA traffic, you know.”

Our building closes at 6 pm, as the receptionist dutifully reminded us around 5.50 pm. The envelope had not been picked up. I was leaving town next morning, and started panicking. Three frantic phone calls later somebody picked up the phone at the company. I explained that the building would be locked in minutes, and was advised to tape the envelope to the front door. At 6.30 pm, I had to leave with a heavy heart.

Next morning I had not received any emails or calls concerning envelopes taped to the front door, so I assumed things were under control. It was in LAX 10 minutes before boarding the plane that the email rolled in: Please log in to see your sequences…

The next few minutes were almost rapturous. I logged in and discovered the majority of the sequences were “great” or “ok.” Few had issues. Knowing that students were using the long weekend to work on their poster presentations and they needed the data, I frantically copied and pasted the sequences into a document and sent them away before boarding the plane.

It was not until late in the evening that I was in internet range again. First I ran a sample I had sequenced before and used as a control. The hotel wifi was a bit sluggish, but Blast finally came back with the correct result: Pseudomonas. I almost hollered.

The sequences came out interesting- some expected genera, others less expected. It will be interesting to see how the genetic data compare with their traditional microbiology tests.

I am happy.

The small details that make love great

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There is a saying in Spanish about “los pequeños detalles que hacen grande el amor.” The small details that make love great.

I was thinking about it today.

Nuclease-free water.

We are doing PCR Thursday as part of the soil project. First PCR reaction in our brandy new PCR machine, and then second electrophoresis run ever in our brandy new electrophoresis set. Laugh if you want. I used to do research in Cuba, using stockings at the end of chromatography columns and candle jars instead of CO2 incubators. Life is good.

I had decided to play safe. No PCR from scratch, let’s get the ready-made beads. For the first trial, we can do with ready-made buffers. Let’s not risk the experiment.

But I forgot the small detail. High quality, nuclease-free water, per the protocol.

We’ll get it by Thursday, somehow. But it is funny, at the end.

We made all those considerations about PCR, ready-made beads and the sequencing place, the ethidium bromide disposal, and the filter to take pictures with the transilluminator. But we forgot the water.

And David Foster Wallace’s commencement speech comes to my mind:

There are these two young fish swimming along and they happen to meet an older fish swimming the other way, who nods at them and says “Morning, boys. How’s the water?” And the two young fish swim on for a bit, and then eventually one of them looks over at the other and goes “What the hell is water?”

Tomorrow will be another day.

Lecture cleanup

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Becoming superhuman?

I happened upon Tim Ferriss’ book The 4-hour body a year ago. I was desperately looking for a system that could keep me more or less fit in spite of the regular academic crunch times. I confess I do not have the discipline of finding some time for exercise no matter what. My tendency is to work on a task basis, which means taking no breaks until done. That meant hours and hours sitting in front of the computer, and it was taking a toll.

The book was fun to read, and the diet and exercise advice worked for me. I do not follow it as rigorously as before, but it is still the basis of how I eat and how I exercise. (Just to be clear: I am not endorsing it or anything, just sharing that it worked for me).

If you are not familiar with Tim Ferriss, he is a sorts of accelerated learning expert as explained in his bestsellers (the latest being The 4-hour chef). In a self-deprecating and humorous manner, he shares his ways of mastering new skills, from new languages to gourmet cooking.

I was thinking about it as I started reviewing my lecture powerpoints for my current microbiology class.

One of Tim’s main points is that most activities can be mastered in an accelerated optimized way. As my teaching takes place in an accelerated environment, most of us teaching at NU explore learning strategies supportive of this approach. Traditional lecturing is not really feasible- many of us combine short lectures with group activities, audiovisuals, or more active approaches to chunk the content and keep students’ interest.

However, as I went over my lecture slides, I started noticing things. Apologies in advance that I am going to talk now about metabolism…that was the chapter I lectured on.

For starters, there were slides that did not make sense where they were except for me. For example, after an introduction to metabolism and metabolic pathways, there was a slide about oxidation-reduction. I know that this is an opening to what is coming next, which is the topic of carbohydrate catabolism. But why talk about oxidation-reduction in abstract first, when I will have to explain it later anyway when referring to what happens during cellular respiration?

The second thing I noticed was lots of material irrelevant for the particular student audience (this case pre-allied health students). Yes, it is pretty to show the names of the components of the electron transport chain, but in all honesty, I do not remember many of them (and I was a biochem major).

Tortora chapter 5

Oxidative phosphorylation, summarized.

I started cleaning up my slides. I took away all information that seemed superfluous, and arranged the slides so there was a clear narrative from beginning to end.

Lecture was yesterday. I followed the narrative of the slides, repeating several times the basic messages. Once students got the relationship between oxidation, reduction, electrons, and hydrogens, I made them look at the metabolic pathway diagrams and count carbons, NADHs, and ATPs. I was stoked seeing their faces, deep in thought as they followed the path of energy. I asked them to turn to their neighbors and explain things with their own words. They asked questions. They were engaged. This chapter is one that I usually dread to teach, but last night was an exception.

We often say “less is more,” but it is not always easy to let go of material or approaches that seemed necessary to achieve something. How much of it is really necessary, and how much of it is tradition and inertia?

Dear educators, how do you achieve a balance between the content that one is supposed to teach and what is really relevant to teach?

Kombucha day 1

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Two starters cultures and tea. Kombucha time!

When I landed yesterday, one major issue was on my mind: my non-majors online General Bio course starting Monday. I had the website more or less organized, but I knew there were things to straighten up before the course shell opened on Sunday. As anybody who has taught online knows, the first week is the most critical: information has to be detailed and easy to find, instructions crystal clear, and instructor responses as fast as possible. So after some grocery shopping and catching up, I spent the night hours trying to tie up all the loose ends.

Two glass containers with kombucha cultures.

West Coast to the left, and East Coast to the right. Same tea. Curious how they will turn out!

In contrast Sunday was a mellower day. Short visit to the beach, coffee, more grocery shopping, and then housework, which these days counts almost as relaxation. Part of housework mixed with science was getting the East-West kombucha challenge. I had obtained a vial of local kombucha some weeks ago, and had it in a vase full of sugary water waiting for the biofilm to develop. Then I had Ashley Shade’s kombucha that traveled with me from Yale. She said it would be fun to compare the two, so I decided to get them started more or less in parallel. Needless to say, while the tea and sugar solution was the same, I cannot really say that the conditions are replicated, as  there is no way to say how many and what type of cells are in the two slimy films that landed in it. But for now my idea is just to get them started side by side. I boiled water and then poured it over the tea leaves (1 tsp/cup). After seeped, I added 1 cup of sugar/liter tea, and let it cool. Once at room temperature, I carefully let the biofilms slide in. Neither floated completely, and I hope they will straighten up eventually as they produce gas. Or not. Covered with double paper towels secured with rubber bands, and placed them on the counter, in a darker corner.

For now I do not intend to collect any samples, just to produce some kombucha and once I have more or less comparable “mothers,” then maybe starting a parallel experiment in earnest.

Oh and if you wonder why are the two containers different, there is a reason.

I could not find two large glass containers of matching size.

Time to visit a Goodwill store 🙂

Home Microbiome- Day 0

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I consider Twitter the social media tool that has been most useful for me. It is not only a real time pulsating (and often distracting) flow of news and information, but also a way to make professional and casual connections. And lately, those virtual conversations have started to show tangible results. From tweetups where you get to meet in real life those people you have been talking to (and often feel you have known for quite a while), to exchange of information, and in extreme cases, to a freezer.

image of a mini freezer

The mini freezer, sitting in the garage

This freezer is part of the Home Microbiome Project, of which you can read here. How did it happen?

Well, of course everything starts with me being an instructor of (among other things), microbiology.  The past years have been exciting in micro, and the fun thing is that the breakthroughs in research have filtered rather quickly to the general public, including students.  I am not sure if it is a general phenomenon related to the availability fo information, or is it that bugs are so interesting- probably a combination of both. The case is that I follow some of the science stuff going on in micro, and one of the threads is the whole microbiome concept. My main interest is still the human microbiota and how it affects health and disease; but still, when I read the word microbiome I usually pay attention. Add Twitter. There are several very active tweeps who are involved with microbiome studies, among them @gilbertjacka, and I became aware of his Home Microbiome project, in which they follow the microbial populations of a home and people before and after a move.  That project sounded really exciting, and when two months ago I got a new position that involves a move, I asked if the study was still going. Not long afterward, a box full with Falcon tubes containing swabs and a bunch of paperwork arrived. Some days later, the freezer made entrance, and was ceremoniously placed in an honorary place in the garage. It has been plugged in for a while, and it seems to work well. 

I am writing this just after midnight. I just activated the gizmos that will record environmental data, and sorted the tubes for early morning’s first swabbing. Two humans and a cat getting swabbed for 6 weeks, every two days. This is so cool.

I guess it is just a geek thing. In my distant past, in places where regulations were not tight, my blood cells became controls of many experiments. Even now, every iteration of a blood lab I volunteer for smears and blood group tests. I have swabbed my wallet, cell phone, and skin at countless micro labs and looked with amazement at the little Staphs growing on the plates.

Anyway, it is day 0 today. The cat has been sneezing, hope he is not getting sick. Hope the microbes filtering from the micro course I will be teaching next week will not upset our microbial ecosystem.

Swab on!


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