Reversing molecular biology: reverse transcription

The polymerase chain reaction (PCR) is not the only lab technique that synthesizes new DNA. In my lab, we often need to make DNA from messenger RNA (mRNA) that we extract from cells, in order to see what kinds of mRNAs the cells are making. Normally, cells use DNA to make RNA—this is called transcription. However, a number of enzymes called reverse transcriptases reverse the process of transcription, making DNA from RNA. (Reverse transcriptases are made naturally in small amounts in many plants and animals, but they are most infamously made by retroviruses like HIV.) Though I expect I will perform reverse transcription reaction sometime, I have not yet, and so I am referencing a Harvard University protocol and a Journal of Biological Methods protocol, rather than my own notes.

Step one: mRNA extraction

Starting with a dish growing the cells of interest, the first step is to extract the mRNA. First, work surfaces and equipment should be wiped with RNase ZAP to deactivate any RNases—enzymes that degrade RNA and thus render the mRNA extraction useless. Use a suction pipette to aspirate—suck away—the liquid culture medium. Add phosphate buffered saline (PBS) and swirl to wash the cells, aspirate the PBS, add more PBS, and use a rubber scraper to dislodge the cells that adhere to the culture dish. After adding more PBS and swirling, pipette the cells into a fresh, conical tube. Now, the cells are ready to work with.

Centrifuge the tube at medium speed so that the cells precipitate down to the bottom, forming a pellet. Without disturbing the pellet, aspirate the medium, then add TRIzol (a solution containing phenol), break up the pellet to return the cells into the medium, wait five minutes, add chloroform, shake vigorously, and wait several minutes for the chloroform and phenol to extract the mRNA, DNA, and proteins from the cells.

Centrifuge, this time at very high speed, to separate the cell extracts into several fractions, like separated salad dressing. The mRNA is in the top fraction, so pipette it into a fresh tube without touching the other fractions, which contain unwanted cell components. Centrifuge again at very high speed for a long time so that the mRNA itself precipitates down into a pellet. Carefully remove the liquid above the fragile pellet and wash it with ethanol. Add more ethanol, then break up the pellet with the pipette tip, and, if all went well, the mRNA is finally in solution.

Step two: DNA degradation

Biology is messy, and any purification procedure, like mRNA extraction, is likely to yield an impure product; DNA is likely to be present in the mRNA. To destroy the DNA, which could cause erroneous qPCR results if it were copied during the qPCR mix the following reagents:

DNase buffer                                                                                                                1.5 µL

DNase (an enzyme that degrades DNA, but not RNA)                                           1.5 µL

mRNA solution (from the mRNA extraction)                                                         12.0 µL

Let the DNase work at room temperature for 15 minutes, then add 1.5 µL of EDTA and heat to 65°C to stop the DNase activity.

Step three: Reverse transcription

The DNA polymerases used to copy DNA during PCR do not copy mRNA. Once the mRNA is purified, it must be converted into DNA by reverse transcription (RT). RT uses the enzyme reverse transcriptase—found in HIV—to synthesize a molecule of DNA from a molecule of mRNA, the reverse direction of normal transcription, in which RNA is made from DNA. First, add 0.5 µL of a primer, then heat to 70°C for ten minutes and place on ice. Then mix the following reagents:

RT buffer (to make sure reverse transcriptase works properly)                               25.0 µL

DTT (to protect the DNA)                                                                                                 10.0 µL

dNTP (the monomers that reverse transcriptase links together into DNA)              5.0 µL

RNAsin (to inhibit any potential RNases)                                                                         1.25 µL

Superscript II (a solution containing reverse transcriptase)                                         5.0 µL

Add 9.5 µL of this mixture to each PCR tube containing the purified mRNA. In a thermocycler, heat the PCR tube to 42°C for 90 minutes, then to 50°C and 70°C for ten minutes to allow reverse transcriptase to synthesize the DNA.

Finally, the mRNA has been converted into DNA, which can be used for several purposes, at least one of which—quantitative PCR, which Jinquan has been using a lot recently to measure gene expression—I will describe in a future post.

Finland – What can we learn?

            Since 2001, the Programme for International Student Assessment (PISA) has ranked Finland’s educational system as the best or among the best in the world, according to “What Americans Keep Ignoring About Finland’s School Success,” a 2011 article in The Atlantic. Americans usually regard a private school as superior to its nearby public schools, a judgement that is usually true, in America. As budgets and teachers vary between public schools, poorer communities tend to have poorer public schools, and private schools are the best option for affluent students; thus, while elementary and secondary education is virtually a right in America, a good education is a wealthy privilege. In Finland, however, even a good education seems to be a right.

File:Northern europe november 1939.png

Paradoxically, Finnish students have performed on par with, but experienced less stress than students in China (Shanghai), Japan, and other countries renowned for education. To begin, education is not compulsory until Finnish children turn seven, according to an article in The Smithsonian, “Why Are Finland’s Schools Successful?” The rationale? “Children learn better when they are ready. Why stress them out?” said Finnish principal Kari Louhivuori. Similarly, Finnish students and teachers both spend fewer hours in class. Students spend much time at recess, which students and teachers value alongside studying; teachers use their extra time to create engaging lessons, and they assign little homework, too. How, then, do Finnish students perform as well as or better than students who study more rigorously?

Perhaps the answer is in part that Finnish teachers are highly qualified, dedicated mentors. Becoming a teacher is competitive; many prospective teachers graduate in the top ten percent of their college classes, according to The Smithsonian again. Americans with a bachelor’s degree in education can teach, while Finnish teachers have been required to earn a master’s degree since 1979; the added credential has made being a teacher nearly as prestigious as being a doctor or lawyer. Teachers, well qualified, respected, and trusted, enjoy a great deal of autonomy in designing their lessons and satisfaction from their work. According to the Center on International Educational Benchmarking, teachers in 2014 were paid between $34,720 (for their first year) and $45,157 (for experienced teachers). If these salaries seem slight compared to the education that Finnish teachers must receive and compared to American teachers’ salaries, which averaged $56,383 in the 2012-13 year, according to the National Center for Education Statistics, note that the ratio of the teachers’ salaries to GDP ranges from 0.89 to 1.17 in Finland but only 0.74 to 1.11 in the United States, again according to the Center on International Educational Benchmarking.

It is remarkable that Finnish schools, from primary education through even graduate schools, are free to attend, with the exception of a small number of private schools. School lunches from primary to the end of secondary school are not only free, but also nutritious, according to the Finnish National Board of Education, which states, “A good school meal is seen as an investment in the future.” Funding for Finnish schools comes from the National government (about 57%) and local governments (about 43%), and even private institutions receive government funding. All schools receive about the same amount of funding per person; schools with large populations of disadvantaged or immigrant students receive extra resources, according to “How the Finnish school system outshines U.S. education,” an article in Stanford News. By contrast, U.S. schools receive about 93% of their funds through state taxes and local property taxes, creating enormous disparities among public schools in affluent, average, and destitute communities, according to the PBS article, “How Do We Fund Our Schools?

File:Finnish school lunch.jpg

Finnish School Lunch, by Vkem on Wikimedia Commons.

It seems that the two roots of Finnish student success are school quality and equality. Quality means that the teachers must be well educated and adept at instruction to become teachers in the first place, that students incorporate both quality class time and play time, and that Finnish schools provide students with nutritious lunches. Equality means that no matter their socioeconomic background, Finnish students all have free access to the same teachers, curricula, and standards because the Finnish national government provides 57% of the funds that public schools receive and because all of the schools draw from the same pool of qualified teachers.

Though these measures have dramatically improved Finnish education over the past 50 years, it does not mean replicating Finland’s educational system in the United States would solve all of our educational woes. Finland, despite its successes, still does not have a perfect educational system, of course; no country ever will. Moreover, it is a smaller country, for starters, and has a different culture and a more homogenous population. However, just as most students learn from their teachers without becoming teachers themselves, the United States can implement and modify policies that have worked in Finland without needing to replicate Finland to improve its educational system.

Malcolm X and the Organization of Afro American Unity

Malcolm X founded the Organization of Afro American Unity because he believed that in 1964, African Americans should not have to contend with racism. Black people throughout all of America, Malcolm X thought, had been attempting to form alliances with each other and with white Americans for much time, without success. Malcolm X reasoned that African Americans would form stronger bonds with each other and with black people in Africa, with “people who look something like we do,” as he put it, than they would with any white Americans. Therefore, he created the Organization of Afro American Unity to allow all Africans across the world to collaborate in order to end racism.

Malcolm X’s call for African Americans to defend themselves is reasonable but seems too vague in drawing the line between defense and aggression. He calls for equality in armament between white and black people, which, once achieved, I would imagine would reduce violence. However, this could encourage black people to aggressively obtain means of self defense. Malcolm X should have clarified how he wanted black people to arm themselves: non-violently, or by any means necessary.

PCR – doubling DNA

Jinquan and I are going to use cells from six mice in future experiments. Some of these mice harbor mutations in a particular gene, and we want to see how that mutation affects the growth of the cells. First, we need to verify that the mice actually have the mutations; otherwise, we would waste weeks working with cell lines that would have the wrong genotype and, consequently, wouldn’t tell us how the genotype affects growth.

Previously, our lab had snipped off a tiny piece of the tail of each mouse, extracted the DNA from the cells, and dissolved it in water. Because we don’t want to keep snipping off tail cells every time we need more DNA, we wanted to conserve the DNA we’d already extracted by taking only a small volume of it—less than necessary to determine the genotypes—and duplicating it using a standard laboratory technique: the polymerase chain reaction, or PCR.

Though I’ve known about PCR since tenth grade Biology, I performed my first PCR reaction this week. Like many procedures, PCR involves following a previously devised formula for pipetting reagents into tubes; it is easy to perform, but you must pay careful attention to what you are doing. Since we had six mice, and I was to put exactly the same reagents—except for the DNA—into the tube of each mouse, I first made a solution that had all of the non-DNA components. Such a solution is called a master mix and has enough reagents to go into all of the individual tubes; I add an extra volume—so seven volumes in this case, instead of six—to make certain that I will not run out of master mix.

Reagent                                               Volume per mouse (µL)                      Master Mix (7x)

doubly distilled H2O                         7.0                                                     49.0

10x enzyme buffer                            1.5                                                     10.5

deoxynucleotides                              1.0                                                       7.0

DNA primers                                       1.2                                                       8.4

Pfu DNA polymerase                         0.3                                                       2.1

Total Volume                                     11.0                                                    77.0

I pipetted 11 µL of master mix into six tiny plastic PCR tubes, and then I added 4 µL of DNA solution from each mouse into a correspondingly labeled PCR tube. PCR mimics in vivo DNA replication. DNA polymerase is the enzyme that actually copies the DNA; the deoxynucleotides are the monomers that polymerase connects to form the new strands; the DNA primers tell the polymerase where to bind to the DNA templates, which I added next. Pipetting small volumes tends to leave drops stuck on the sides of the tubes, so I pulled all of the liquid down from the tubes’ walls by centrifuging them for two seconds.

I then placed the tubes into a machine called a thermocycler. The thermocycler first heats the DNA to separate the strands, then cools the mixture to allow the primers to attach and polymerase to synthesize a new strand on each extant strand, and then cycles between heating and cooling, doubling the DNA each time. With Jinquan’s instructions, I programmed the thermocycler’s temperature and time settings, then let the reaction run for 35 cycles. Theoretically, the amount of DNA doubles during every cycle, so we should be left with 235 times as much DNA as we started with. This is enough DNA to allow us to visualize it on a gel, which will be the subject of a future post on what it is like to run a DNA gel.

It’s the Scientist that Counts

You’d think that counting things would be easy by now. My first grade class practiced counting these red and yellow chips called “counters” twelve years ago, and I assumed that counting cells would not be so much different.

Counter Pic

Two-Color Counters – EAI Education

Well, counting cells is not so straightforward a process as was passing time with counters in first grade. My purpose in counting was thus: Jinquan—the graduate student with whom I am working—had been growing U2OS cells—a type of bone cancer cell—in six-well plates, and we wanted to measure the number of cells growing in each well to determine wh  ether or not the absence of a certain protein was affecting their growth rates.

In a six-well plate, there are two to three milliliters of medium per well, and each milliliter contained between 100 thousand and one million cells per milliliter—not so easy as counting ten counters. We used a device called a hemocytometer—literally a “blood cell measurer”—and a microscope to count the cells in a very small volume—100 nanoliters—and then multiplied by ten thousand to estimate the number of cells per milliter. It all goes well, unless different samples of the same cells start yielding radically different results, which happened to me.

Hemocytometer

Hemocytometer with a gloved hand – Jeffery Vinocur

There are two different methods I learned to load a hemocytometer. The first is to place a coverslip on top of the smooth platform in the center of the device, creating a 100-micrometer gap between the platform and the coverslip, and then to pipette ten microliters of cells into the grooves, such that the cells are pulled by capillary action into the gap. The second method is to pipette a drop of cells onto each platform, touch a coverslip to the top of each drop, and, keeping the coverslip perfectly level, lower it onto the hemocytometer. The problem was that using the first method, I counted one fifth the number of cells that I counted using the second method. Moreover, my counts disagreed with Jinquan’s. In order to improve my hemocytometer skills, Jinquan emailed me a video on the proper way to use the hemocytometer.

After watching the video, I was still puzzled by the discrepancies between the numbers of cells I counted using the different methods, but I decided that I needed to ask Jinquan about the rules we follow when counting cells in our lab.

For example, when you look at a hemocytometer under the microscope, you see a grid over which many cells are dispersed, count the number of cells in specific millimeter-by-millimeter squares, and then take the average. But what if a cell lies on the boundary between two squares? Jinquan told me to count cell on the left or top boundary but ignore those on the bottom or right boundaries. And what if I can’t tell whether or not something actually is a cell?

Most of the time, distinguishing a cell from a non-cell is easy: cells have thick borders and somewhat resemble glass beads. But distinguishing small cells from non-living specks—which are called artifacts—is not so straightforward. Jinquan told me that if an object looks flat, diffuse, or much smaller than most of the other cells, it is probably an artifact.

Cell Squares

Cells on a hemocytometer – Joseph Elsbernd

Though this guideline helps me pinpoint most artifacts—those three small dots in the top row of the bottom-right-most full square are unequivocally artifacts—but still, there is a threshold of uncertainty on which sit some objects, such as the faint one in the third row and first column of the aforementioned square. Is it a cell? I would count it, because it seems just large enough and its border is well defined, albeit faint, but I am not certain.

On Monday, Jinquan will load U2OS cells into the hemocytometer, and I then will load it using cells from the same tube. She will count them, and then I will count the exact same slide to see if our numbers agree. We will elucidate the cause of the discrepancies between our counts and the internal inconsistencies within my counts and thereby become confident in our data and in my ability to accurately count cells in the future.

Free College?

Does two free years of college sound too good to be true? President Obama plans to make community college free to all half- and full-time students who maintain GPAs of at least 2.5, which he announced on January 9, according to the Washington Post article, “Obama announces free community college plan.” Obama supported his plan by saying that “America thrived in the 20th century in large part because we made high school the norm,” and that these days, “A college degree is the surest ticket to the middle class” and “ensures you are always employable.”

Using unemployment data released in January 2015 by the Bureau of Labor Statistics, I calculated that from August to December 2014, the average unemployment rate among high school graduates without college education was 5.6%, but was 5.1% among those with some college or an associate’s degree (the type of two-year degree awarded by community colleges), and, by performing a t test, I found that the difference was not due to chance (p = 0.039). However, during the same time period, average unemployment among those with bachelor’s degrees was only 3.0%, significantly (p = 0.0001) lower than unemployment among those with an associate’s degree or partial college education. Thus, it is incorrect to say that an associate’s degree ensures employability, but the data show that people with even an associate’s degree are less likely to be unemployed than those without college experience, though more likely to be unemployed than people with a bachelor’s degree.

Unemployment statistics alone do not consider incomes. According to the National Center for Education Statistics, the median 2012 income of those with a bachelor’s degree was $46,000, but only $36,000 for those with an associate’s degree. However, for high school graduates with no degree, it was $30,000. The immediate cost of not being employed while attending community college for two years would thus be around $60,000, but the average community college graduate would recuperate the difference in about ten years, and so the data show that for an individual, earning an associate’s degree for free would be worth the time.

What about the effects of Obama’s plan on broader society? While free community college has not been tested in all of the United States, some states have such programs. In California, according to the January 28 Washington Post article, “Obama’s free college plan is no panacea; just ask California,” about two thirds of full-time community college students have their tuition waived. However, the article cautions, by offering free education, community colleges must accommodate more students and spread their resources more thinly to serve them all. Currently, only about 50% of those who enroll actually finish their degrees, but if future students can use fewer resources, that percentage could drop.

Education that is free for students naturally raises the question, “Who pays?” The aforementioned “free community college plan” article estimates that 9 million students will participate and cost a total of $6 billion per year for the next ten years. Obama has outlined, but not detailed, plans that would increase taxes for the wealthy, according to this New York Times article. These plans include eliminating the trust-fund loophole (whereby wealthy people can buy assets that accumulate value over their lifetimes, and then bequeath them to relatives without being taxed on the amount of accumulated value), increasing the capital-gains tax rate from 23.8% to 28% for families earning over $500,000, and imposing new taxes on large banks and financial firms. Meanwhile, Obama wants to cut middle-class taxes, instituting a $500 tax credit for families with two working parents and up to a $3,000 credit for each child under 5. It is estimated that tax cuts will amount to $175 billion and tax revenue will be $320 billion over the next decade, which would help fund Obama’s free community colleges.

If the plan goes through and works, that is. Many people, especially Republicans, oppose the plan, and given that Republicans constitute the majority of Congress, passing it may prove difficult. Utah’s Republican Senator Orrin Hatch, for example, said that increasing taxes on businesses and investors would undermine the successes of previous tax policies.

If Obama’s plans do pass, they beg the question, “What will happen to United States education in coming decades?” The average number of years spent in school has been increasing over the past century, and if ever-higher levels of education become ever more accessible, the percentage of people with advanced degrees will continue to rise, so jobs will become more exclusive for people with lower degrees. It will be ever harder for people with associate’s degrees to find jobs, harder for people with bachelor’s degrees, and harder for people with master’s degrees. Most jobs do not require a Ph.D-level of education, and if we got to the point where one needed a Ph.D. to become an electrician when an associate’s degree or simply an apprenticeship would be perfectly fine, then the government would be wasting many resources on education, and people would be wasting many years on it. Determining the “right level” of education is beyond the scope of this post, but I plan to address it in future posts. Hopefully, Obama’s plan for free community college education will allow at least half of Americans who should have a degree but cannot afford tuition to attend college and learn the skills they need for their careers.

Blog Topics Revealed

For my Passion Blog, I have decided to write about my experiences with doing research in Dr. Wang’s gene regulation laboratory since September of 2014, for four purposes:

First, I want to help inform those of you who are interested in research but either haven’t started or are working in an area that is very different from my own area of research. With knowledge of what working in a wet lab and dealing with pipettes, cell cultures, and experiments that don’t work the way you wanted them to, I hope that you can make more informed decisions about what kind of lab to join.

Second, I want to show those of you who are not interested in research what working in a lab is like so that you do not continue to think of lab work, if you think so now, as some kind of black box accessible to mad scientists in white lab coats. (I haven’t actually yet worn a lab coat in the lab; go figure!)

Third, I want to investigate certain laboratory procedures that I myself will do in the coming months, such as western blotting, and write about them so that I will know in advance how to perform them.

Finally, I want to improve my skills of translating scientific jargon and techniques into text that the average non-science major can understand. I believe that everyone should know how science works and should not be overwhelmed, thinking that they have no hope of understanding. The only scientific thing people have no hope of ever understanding is quantum mechanics. (I say as a biochem major.)

This quote is probably misattributed to Albert Einstein, but it follows: “If you can’t explain it simply, you don’t understand it well enough.”

 

For my Civic Issues Blog, I would like to write about the controversies surrounding education. I believe that education is our hope of a good future, but there is no one right way to educate a student, and thus I would like to investigate many issues, including:

How does one reliably measure student performance?

How does one reliably measure teacher performance?

What approaches to education, such as teacher-lectures-students-listen (direct instruction), differentiated instruction, home schooling, autodidacticism, and cyber schooling seem to work best?

How does learning become fun, not stressful?

How can teachers make students genuinely interested in the material?

How much time spent in school is wasted? How could school time be used more efficiently?

Is college tuition going to continue to rise, and how will students afford it if the increases in their eventual salaries cannot keep pace?

And how should we reduce illiteracy in countries where it is rampant, such as how the Malini Foundation aims to educate women in Sri Lanka?

“This I Believe” more coherently

Two people work on the same London double-decker bus. One drives the bus. The other walks around collecting passengers’ fares all day. Who is more likely to die of heart disease?

We have known the answer for sixty years. The renowned epidemiologist Jerry Noah Morris researched this question in 1953 and, not surprisingly, found that the sedentary drivers were significantly more susceptible to heart disease than were the ambulatory conductors. The same bus, the same hours—let’s even for now assume the same salary—but not the same rate of heart disease. And so, if you were the driver and were offered the conductor’s job, would you accept that substitution?

I believe that the simple substitutions we unthinkingly make, day in and day out, determine our fates. And that is why I stand up in class. I have been standing for almost two years now—two years of exercising my soleus muscles instead of letting them dangle. Now, a common excuse for not exercising, and one for which I have been guilty, is a lack of time. But I know that standing up in class takes exactly as much time as sitting, yet look at the long-term benefits! I have no bloodwork data for myself, but innumerable research studies have found that standing more and sitting less is associated with lower insulin spikes, lower cholesterol and triglycerides, and lower risk of death. All by one simple substitution.

Now, take standing on the job a step further—or maybe ten thousand steps—and voila, the treadmill desk. While the benefits of standing are largely invisible, the benefits of walking are countable data—by mile or by step. While writing his fourth book, Drop Dead Healthy, A. J. Jacobs walked twelve hundred miles on his treadmill-laptop desk, substituting it for the sitting desk where he wrote his first book, The Know-it-All, when he probably just walked to the bathroom.

I too made walking habitual towards the end of tenth grade, walking to school instead of riding the bus. To walk one way took twenty minutes, and to ride took ten, but I regarded those twenty minutes as wholly productive, and those ten as sedentary waste. On days I walked both ways, my pedometer clicked five thousand extra steps. And so, by taking only twenty extra minutes each day, from the end of tenth grade until the end of twelfth, I substituted nearly eight hundred walking miles for eight hundred folded in a cramped, yellow bus.

I believe that many maladies that exist today—heart disease, obesity, and such—result in part from substituting comfort or convenience for salubrity. But I also believe that by pushing ourselves outside our comfortable niches—by substituting back the healthy behaviors we were meant to do, we can reverse these epidemics. Will that be comfortable? The first two weeks I stood in school, explaining what in the heck I was doing embarrassed me. My calves and ankles grew stiff and sore. But the pain did subside. And there were many days—frigid, blustery days—when I yearned to be seated in a heated bus as I pushed against the wind. But I kept walking.

“This I Believe” before editing my belief.

Twenty-four. Tick. Tick. Not enough time—never enough for us. And yet, how do we explain why it is enough for prolific writers and seminal ? How enough time for Wolfgang Amadeus Mozart to write 625 compositions. Let me add that Mozart died a month before turning 36—he lived fewer hours than I hope any of us will, yet is among the most prolific of composers. How?

Wolfgang Amadeus Mozart didn’t watch Teletubbies or play World of Warcraft. At age three, he followed his sister Nannerl to the harpsichord bench, where his father Leopold ensured he practiced hours per day. At age five, he began composing, and by age ten, had published his first sonatas and symphonies.

Why didn’t we all publish music before we hit puberty? We had the time. We have 24 hours each day, the same as Mozart. The same as Archimedes. The same as Gandhi. And the same as Benjamin Franklin, who—amid printing newspapers, studying electricity, and leading the American Revolution—had the time to record how he did those things.

Here is a quote from Franklin’s autobiography, just after he had begun working and eating at a printing press in London.

“I drank only water; the other workmen, near fifty in number, were great guzzlers of beer…. My companion at the press drank every day a pint before breakfast, a pint at breakfast with his bread and cheese, a pint between breakfast and dinner, a pint at dinner, a pint in the afternoon about six o’clock, and another when he had done his day’s work. I thought it a detestable custom; but it was necessary, he suppos’d, to drink strong beer, that he might be strong to labour. I endeavoured to convince him that the bodily strength afforded by beer could only be in proportion to the grain or flour of the barley dissolved in the water of which it was made; that there was more flour in a pennyworth of bread; and therefore, if he would eat that with a pint of water, it would give him more strength than a quart of beer. He drank on, however, and had four or five shillings to pay out of his wages every Saturday night for that muddling liquor; an expense I was free from. And thus these poor devils keep themselves always under.”

End quote. I am then not the first to believe that the simple substitutions we make determine how successful we are. Simply choosing water rather than beer determined how successful Franklin was. Simply relieving himself of the need for beer’s taste and strength relieved Franklin of paying for it. It relieved him of cloudy intoxication, such that Franklin could efficiently use his 24 hours. Simply saying, “I don’t need this comfort, or this luxury, or this idle time; there is something better I can do,” I believe we can all make simple substitutions. I believe we can all replace our spades with climbing picks. That we can stop digging holes, one scoop per day, and start ascending the mountains where we really want to be.

A Note or Two on Research

Do you want to work in a research lab? I did as well at the beginning of last semester; actually, I’d wanted to work in a research lab since eleventh grade. I read up on lab techniques in my AP Biology textbook in twelfth grade, and it seemed that performing basic lab work would be neat and simple—you know, just mix this enzyme with this plasmid (a small, circular piece of DNA, often found in bacteria like E. coli), wait 30 minutes, transfect the mixture into bacteria, and let them grow. It also seemed like it would succeed most of the time—I mean, biology is a science, and being a science, it must be able to be repeated, so if one enzyme-DNA reaction works, they should all work. I was completely wrong.

500px-Plasmid_(english)

Plasmid figure posted by Spaully under Creative Commons License. Link.

I’ve made a lot of mistakes, and for those of you who are interested in joining a research lab, I’d like to share with you my mistakes, my failures, and my misconceptions, as well as my successes—don’t misunderstand me, my efforts sometimes work—in hopes that when you join a lab, your understanding of what lab work is like will be superior to my understanding of it back in September, when I joined Dr. Yanming Wang’s gene regulation lab.

So here is one of the first things I learned about research, and it’s probably the most important lesson I learned last semester:

Take notes that are sufficiently detailed, such that, if someone wiped your mind of the work you did, they would tell you how to repeat every single step exactly.

For the first few weeks of looking over the shoulder of one of the grad students in Dr. Wang’s lab, I typed my notes into an Excel spreadsheet, and a typical day’s notes looked like this:

Date Time in Time out Notes
24 Oct 2014 9:10 11:05 Following the protocol for extracting plasmids: centrifuging out E. coli, aspirating the medium,  resuspending the cells, lysing the cells, precipitating out the large components, pipetting off the DNA remaining in solution, putting it in a tube with a DNA-binding matrix, collecting the DNA in the matrix, and eluting the DNA off from the matrix.

Basically, every procedure that one does in the lab is called a protocol. What I was taking notes on were the general steps of the protocol for extracting a plasmid from E. coli. What I should have been taking notes on were the detailed steps:

  • For how long did I centrifuge out the coli, and at what speed?
  • What did I use to aspirate (that means to suck off using a vacuum) the medium?
  • What did I add to the coli to resuspend the cells?

And so on. Now, I hand-write all of my notes on loose-leaf paper, and they look like this:

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The notes above are also for extracting a plasmid, but rather than say, “Resuspending the cells, lysing the cells [that is, breaking open the E. coli cells so that the plasmid DNA we want spills out]” and so on, they read, “Add 250 µL [a µL is a microliter, a millionth of a liter] of Soln. 1 [solution 1], pipette to mix [to resuspend the cells], add 250 µL soln. 2 [to lyse the E. coli cells], invert 6x, wait 3 min,” and so on. Every step in my detailed notes corresponds to one in my cursory notes. The difference is that the cursory notes tell me what the result of each step is, but the detailed notes tell me exactly how to perform, and, if necessary, to repeat each step using solutions 1 and 2 from the plasmid extraction kit.

By taking detailed notes, I can look back at my notes and quickly see exactly what I did on a given day, which helps me quickly resume working and save time. It also lets me detect any mistakes I might have made in the details, which I would not have recorded with my former, cursory note-taking style. And, as taking notes helps me learn the material covered in regular classes, taking notes helps also helps me learn the protocols in the lab. Therefore, once I figured out how to take better notes, I was ready to learn how to perform the most vital tasks in my lab. They will be the subjects of my subsequent posts.