My Reflection on Naturally Obsessed: the making of a scientist

Naturally Obsessed: The making of a scientist is a documentary-like movie filmed in the special and rarely seen realm of a working molecular biology laboratory in Colombia University. Three student scientists are the storytellers’ of their research experience mainly in the laboratory and even in their non-academic, social lives outside out there. As a fellow science student (future scientist), I will stick to these three students of Larry, instead of him in particular. In general, all of them have most descriptions about their academia in common as they are studying on the same field. But this necessarily does not mean that they hold fairly similar opinion or passion for the intensive research they are spending most of their time on. Starting from their initial thoughts and self-commitments in encounter towards the Ph.D. they all are in for, their background life stories before getting to this present time (i.e. the time of the filming) and several factors in their ways of critical thinking and approaching problems are major separating points among the three.

To begin with my favorite character, Rob, is a very unique and interesting individual. I believe anyone can tell that he has the potential to do great things like what he does throughout the movie. If you are one who has noticed that and agrees with this statement to some extent, guess what these qualities of a good scientist in Rob are derived from? You may have a different point of view or even disagree with me at all but I strongly believe that almost everything that Rob does in the laboratory is a shadow created by the light he has ignited at the very beginning of the research after he drops out for three months, thinks of quitting, but returns to the lab. I think that has been a great turning point in his life as he tells us “[T]here's this itch or this anxiety that you have that can only be supplied by knowing” about scientific research. He has also clarifies that receiving his Ph.D. according to his astonishing accomplishment of his research is the one and only promise that life is still keeping for him. He has burned down all the bridges but only this one. So it is not a secured and safe trip for him to risk anything major. He falls down a couple of times in the past but he is back in the game now and rising up higher than any of the ditches he has been under. He understands and interprets his results as much, if not better than his colleagues in the laboratory.

Second, Gabrielle is a model student scientist who has worked as a lab technician in a university right after her graduation and has decided to attend a graduate school as she has not been satisfied with the quality of life and fulfillment of academic starvation she has. According to her principal investigator and her colleagues on the movie, she is a pretty decent individual and fellow scientist. One of the many great things that she has brought up in the movie is the beauty in the cycle of research as she has mentioned “[M]aybe today I'll have the answer to this question and then I'll have more questions that I'll be able to ask.” She has a noticeable weakness as she analyzes the situation that she hasn’t been aggressive enough in getting help from her PI, Larry. Unfortunately, it was very heart breaking to watch that her dropping out from Colombia to start working for a bio tech company. On her interview after she has been gone away from the laboratory, she has still proven herself as she explains her reason on dropping out indirectly by telling us she is happy with her new profession as has replaced research with productivity. Personally, I do not quiet agree with her point on that as that productivity she refers to is an offspring of a research that someone else has done in the past. Actually, I strongly believe the world rotates around science research and most of all and especially, biochemistry rules at all times (This is my blunt opinion).

Lastly, Kilpatrick is an extremely dedicated scientist who has an outstanding skill of reasoning and discussion. This can be well-proved in the movie even by just visualizing him having active conversations with different people throughout. He has also mastered the ability in becoming a great listener too. Sometimes, listening to others is much more advantageous than speaking one’s mind while just hearing but not listening. Kil has inspired me as he has sacrificed the most by breaking up his engagement and prioritized his research over his fiancée. Although he has nothing guaranteed about receiving the Ph.D., he has eliminated a cause of distraction from his limited and precious time (I do not infer to his fiancée at all but the frequent fights that they were having about him taking too long to graduate from what he has told us in the movie). This a magnificent real-life example that portrays that a research is a very challenging and demanding process that might definitely feel exploitative and ridiculously bitter along the way; but it always has  an endless nectar in it in the meanwhile which should keep you interested and energized if you would give it your mind and heart. No matter what would happen to Kil, he has gathered all that priceless knowledge from his tremendous days and nights in Larry’s lab.

Literature Cited

Squires, Buddy, dir. "Naturally Obsessed | THIRTEEN." Naturally Obsessed: a Making of the Scientist. PBS. PBS, New York City, NY. THIRTEEN - New York Public Media. Web. 20 Jan. 2011. <http://www.thirteen.org/naturally-obsessed/>. Transcript.

"Naturally Obsessed. The making of a scientist." Naturally Obsessed. 18 Jan. 2011. <http://naturallyobsessed.com/>.

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My Reflection on Some Perspectives and How to Succeed in Scientific Research

            Martin A. Schwartz’s journal of cell science, The importance of stupidity in scientific research, has made me laugh and feel so interested to question its publicity according to the title before I have started reading it. Actually, it is not a good approach towards reading a piece when a reader prejudges it without a detailed look at its content. Anyways, I have read the journal and my perspective on it is totally changed now. It is such a great work to read and I have felt ashamed to think of it as a meaningless writing. During my accomplishment of reading this well-written journal, I have learned a major feature of being a researcher in a scientific research project, especially as a graduate school student, which is the researcher’s feeling stupid throughout the research. This is a bizarre concept which might not make any sense to a reader of this journal that does not have any base of be involved in scientific researches. But for me, being a science student who is working on a research project, I can definitely relate my experience in the laboratory with Schwartz’s situation. I always feel stupid and unsatisfied at the end of the day in the laboratory and that sometimes can even get to the point that I partially lose my interest in the project as I get so much discouraged. I believe that this feeling is mainly originated from the repeated negative results of my experiments. One thing that I cannot deny in this whole scenario is that I am getting so much knowledge from my ‘failures’ and ‘stupidity’ from each of those days. Obviously, learning is the climax of any student’s goal in attending an institution and that’s what a graduate school student achieves everyday he/she feels like dumb as a rock. That friend of Schwartz’s who is a lawyer now is a bright woman to identify that science is not her strongest field of studies but law instead; but I also want to point out that she is also not the most convincing and successful lawyer out there as she could not take the challenge of graduate school and dropped out. In my personal case, I want to extend my circumference of studying onto a medical school in my career. Medical school is very similar to research graduate school in several aspects and the type of courses offered and their context are the most challenging characteristics they have in common unlike high school and college courses. In general, I will always keep Schwartz’s piece of advice in mind like the powerhouse to the survival of my career and goal in becoming a well-recognized and ‘knowledge-rich’ health science professional thanks to my stupidity in my scientific research of today.          

            Jonathan W. Yewdell’s essay How to Succeed in Science: a concise guide for young biomedical scientists has two parts that deal with different aspects of a research. In Part I: taking the plunge, Yewdell has brought up very important issues and satisfactory responses to most of the questions that young science students, like me, have posed. It is basically a map of the graduate and post-graduate schools. I strongly believe that a successful and passionate science student who has a science related career should know this standards and rules/regulations about higher level education after undergraduate school as important as a good camper should carry a map or compass along on a trip no matter how much experienced and familiar he/she is with a particular route ahead of him/her to reach a destination. Yewdell has well-specified the requirements, recommended inclusive, ‘never do’s, irrelevant points and several possible alternatives that could be taken within this trip. This trip in the essay starts from the time of applying to the very last miles before the destination like the ways to become an independent professional up to the climax of becoming a principal investigator (PI). Indicators of the field of studies or graduate school as in whole not being the best fit for one is not hidden in the essay either. The student’s rights to make the decisions in managing the trip to his/her career goal are also specified. Picking a mentor and a laboratory are pointed out being very important parts of the whole experience as they are known in playing a key role in the students present and future achievements in his/her career. As of graduate schools, this Intensive Science Research course is a very crucial way of understanding the concepts carried by this essay as each of us spend around 35 hours of laboratory work per week, attend seminar meetings, shadow each other and discuss our projects, present and teach our colleagues, keep laboratory notebooks and several other things. Specifically in my case, everything does not hold the same for me and my career plans because medical school has a different approach and requirements throughout the processes before, during and after attending compared to graduate school. As the essay continues, in Part II: making discoveries, Yewdell extends his coverage about scientific research as he discusses and mainly advices his readers on the progress of the research starting from choosing the project to the biggest essence of most, if not all, projects: discovering results. Various objectives of the different stages of a scientific research are discussed and some disciplinary and safety issues are also parts of this piece. The experience in the laboratory is, and definitely should be, something bigger than the studies that one does and learning scientific things only; but it includes the friendship and fraternity that develops among the young scientists (i.e. the 11 0f us in this course). Not enjoying the laboratory environment and those who are around you definitely influences your outcomes and results of your experiments to some extent. Although it might sound almost impossible, all of us have to have fun in the laboratory. I actually enjoy the laboratory environment a lot and I do not mind staying there and sharing my findings and knowledge with my colleagues.

Literature Cited

Schwartz, MA. The importance of stupidity in scientific research. J Cell Sci. 2008 Jun 1; 121(Pt 11):1771.

Yewdell, JW.  How to succeed in science: a concise guide for young biomedical scientists. Nat Rev Mol Cell Biol. 2008 May; 9(5):413-6.

Changes in my Opinion on the Intensive Research

            The intensive research is changing my opinion of how research is done and what it would mean to be a scientist doing research as a career. I have a positive perspective about the overall idea of research and I know that it is significant, if not the most effective, way of learning and understanding a concept. As of any other things, it also has its own strengths and weaknesses. One can definitely argue that research does not play any role in the learning environment; but I strongly disagree with that. Research, especially and specifically, science research is the key to develop the sympathetic of the world around us. We all know completely nothing when we were introduced to the community we belong in and we have learned each and everything that we do in our daily lives gradually as we grow up within that community. It starts from things as simple (i.e. for most of the adult human) as walking and speaking. This phenomenon keeps on growing and developing gradually throughout the years and great achievements of the mysterious human brain and its interactions with other factors and individuals around it has reached since its creation and/or evolution from its ancestors. But, I want to point out the logic that this does not quiet happen just by the personal experiences of each individuals’. Knowledge has been flowing among generations in one way or the other. And this knowledge has to be gathered, supported and /or disclaimed by a research; especially, a science research.

            When I have started my research project in the fall of 2010, I was a new born child to the bimolecular and biochemistry world. I have had a great interest in knowing several techniques/protocols and understanding the minute details of how the nucleic acids function and all the other possible sub-sections of this field the study. I have known that getting engaged in a science research and working with these subjects being a first person myself. As a result, I have decided to start working in the DNA lab for an independent research study to have the experience of it. I have had a mixed feeling about it: interested and curious but confused and scared. I was not sure if everything would be flowing smoothly and positively or in a frustrating and surprisingly unexpected way. Anyways, it is called research because I have to find out all of these questions at that point of time answered by just doing it and learning each and every aspect of it. So, I have convinced myself to do it and keep on going forward with it, never give up or feel discouraged since nothing in the research is supposed to happen in a certain way always (I can say that now since I have learned my lesson). In general, my opinion about research in general was a distorted image for some reasons that I cannot tell how they have developed and why.

            After having a four-month-long experience of it and doing fairly for quite a while, I believe that research is a process of a chain of learning new things no matter how the results end up like or if it matches what I have planned in my hypothesis at the beginning. It is so great because it is always a win, win situation in which you still learn a thing or two out of every finding that your research would drive you into. With that been have already said, I used to think that independent research has meant by literally independent and left alone kind of sad situation; but guess what, once again I was wrong. Yes it is independent in the sense it is me who is the one who does all the active learning part and all the hands on experience but what I was missing was the huge picture behind all my studying experience in the lab: my mentor. It is a pleasure to work with one of this highly dedicated and devoted people who have already done most, if not all, of the procedures that we follow at some point in the past being a graduate school students or scientists. They are most definitely important sources of knowledge and ideas who are always next to us in our/my journey in the research experience. So, this also made me to come to the understanding that planning and brainstorming before the progress of the lab procedure and discussing and reflecting are also a very major, important aspects of the research experience. I actually have had a slight concept of this even before starting to work at my current research but I have not understood that it should also be done with other students who are studying similar and different fields and mostly my mentor. I believe it is one of the main reasons for having the mentor so that we can do the checklist method or some other related things with to ensure that I am on the right track. Shadowing others and learning and interacting with others is the other option of getting the best out of this research experience and making it more interesting. I believe everyone has limited amount of knowledge and applying that as the only way to approach a problem would not last for that long or make you reach that far; instead, diversifying those great ideas in each individual would make the overall ending much thoughtful and advantageous for the whole science community that is working independently but also together. This part of the research experience even glorifies the hidden image of the power of  the research in making a society and develops fraternity among those students/scientists both and/or either inside and outside the laboratory area. So, these and other some points are the changes in my understanding of how research is done.

            Finally, to be a scientist doing research as a career, in my opinion, means, first of all, determining one’s self for lots of exciting experiences and time committed into the laboratory. Obviously, the scientist’s expertise and credibility in the specific field of study that he/she is involved in would get much better and more trustworthy. In the meanwhile, the rest of the world and other scientists would be interested in getting to know and understanding the results of that scientist’s studies from his/her experiences so he/she would be invited to attend several conferences and seminars to learn more, share his/her achievements, get others opinions (i.e. such as in by shadowing) and suggestions for other possible alternatives to consider for their further studies in the search for better results. The world and most of the different communities in it are highly interested in finding the solutions to the major issues they are dealing with in their daily lives; so they/we are in so much need of as many strong-minded scientists as possible, not super heroes.                       

My Shadowing Experience

            As a science student who is involved in research studies, I have extended my horizon of scientific understanding and learning by shadowing a colleague who is engaged in a totally different field of studies. I have got the opportunity to shadow Cara Marie Voelliger in her experiment on fruit flies, Drosophila melanogaster, to study how they get affected by the presence of different concentrations of cadmium in their environment. First of all, the fruit flies are chosen as the subject of this study as they are the most common and important model organism in modern biology. To do this, she has run numerous, long term trials of observing the characteristics of a virgin male and a virgin female fruit flies in each of several vials which contain the different concentrations of the cadmium. In this experiment, Cara is mainly looking for the copulation/mating of the flies in the vial. The length of each observation lasts for 30 minutes. I have noticed that it is a really interesting topic to study but, in the meanwhile, it also can get so boring and frustrating as the scientist has to focus on it for that time interval and gives each and every move of the flies an undivided attention with highly crucial timing of the specific activities.

            Other studies that have been done in the past imply that heavy metals (i.e. including cadmium) have a non-linear and fluctuating effects on the behaviors and sexual orientations of these organism. According to these studies, fruit flies could easily get stroked by the application of low and high concentrations of the heavy metal; but, surprisingly they do not recognize the presence of the medium concentration. There are no justifications and reasonable explanations to this phenomenon yet. This is not what Cara is also trying to investigate. It helps anyone to understand and accept the possible results though. In general, heavy metals, most likely, affect the fruit flies by varying the amino acid in them called methionine. Some studies have shown that dietary methionine can modify lifespan in adult female, fruit flies fed a synthetic food with carefully controlled amino acid, sugar and nutrient content.* This actually has nothing to do with Cara’s research but it implies another characteristic feature of the amino acid within the same organism used as a study subject in her experiment. But it might just open a door for another research and I believe that is not Cara’s interest at least for the time being.    

            Currently, Cara is working with 0.75 mM cadmium which is the highest possible concentration that would allow the fruit flies to stay alive for the research. So, it is most likely that this concentration has a negative effect on the fruit flies as it is close to becoming deadly against them. She has also tested 0.1 mM cadmium in the past and her observations report that the flies were negatively affected by that concentration. In the other hand, a medium concentration, 0.5 mM, cadmium has not shown any significant difference from the wild type flies. Therefore, even if we cannot tell the reason, low and high concentrations of cadmium have negative results while medium concentration of cadmium has no known negative effect so far.

            Supported by the theory and background studies that have been mentioned above, Cara is not seeing any copulation in any of the 0.75 mM cadmium vials. There is actually unlimited number of possibilities for the results to come out this way in addition to the toxicity or hallucination that the cadmium might have been causing to the flies. Ether is also a major part of the procedure in this experimentation and over-ethering the flies might have a long term effect on them so that its effects on the behavioral changes of the flies might be expressed later during the time of the observation.

The length of the time of copulating is another factor to be tested in this laboratory set up which is actually being done as a side support of the observations. As different heavy metals and different concentrations have different effects on the behaviors of the individuals which are involved in the vial, every factor should be tested and the degree of the influence should also be known to the possible depth. During her observations, Cara looks for minute details that might be done by the flies such as: preening which is the cleaning up process by a fly, singing to attract the female, sight and chasing after the female around the vial, touching/tapping the female, and all the other several suspicious activities for copulating and ‘normality.’ Specifically, any type of contact has to be awarded a major attention by the scientist as it is not any simple physical contact happening between the two but it is most likely a chemical transfer by the male via its extended proboscis to lick the female’s genital area and trigger sexual feelings which would allow its dear request for mating instead. In this experimental community of the two, the female has a total presidency over the male when it comes down to copulate. It is all up to the male to seduce the female in any possible way to mate with.

In comparison to my research, this research that Cara works on has very few similarities and several differences to mine. To start with the similarities, both of us are holding some previously done research results as our background information and we both have that checkpoint to come up with a critical thinking approach of tackling the problem that we are trying to solve in our research projects. The various variables, especially the controls that we have included in our studies' also make our research projects' somewhat similar as we both have something to use as a point of reference. On the contrary, our differences start from the fields that we are researching: Cara's behavioral science and toxicology versus my cell and molecular biology. I deal with micro studies while she deals with the bigger picture. I would also say that Cara has some specific hypothesis that she has based research on while I have a goal as most researches do but I would not certainly say that I have a specific hypothesis to relate my research to. These are the very few similarities and differences that I have noticed during my shadowing experience. 

 The involvement of the human emotion from the scientists’ side is likely in behavioral studies such as this one. Most biologists argue that it should be eliminated as much as possible as it would create bias into the observations made and then the results as well. Basically, it is difficult and almost impossible to eliminate one’s emotion and thoughts but it is also a great thing to understand and accept the truth that this experiment has subjects which are not humans, even if they have minor similarities, so that we cannot and should not treat them or interact with them as of humans. Therefore, we should not expect the flies to act in a certain way or react to a factor in any types at any time throughout the experiment; as none of us can do that. This actually reminded me of the article The Brief history of the Hypothesis that I have written a blog about last week when it comes to the point about how our assumptions might influence our results. They would possibly change the results and findings of the experiment to any extent. The solution, in my opinion, is to eliminate them as it is almost impossible to exclude them being a human.

Literature Cited

*Troen, A.M., French, E.E., Roberts, J.F., Selhub, J., Ordovas, J.M., Parnell, L.D., Lai, C. 2006. Lifespan modification by glucose and methionine in Drosophila melanogaster fed a chemically defined diet. Age.29:29-39.

To Hypothesize or Not to Hypothesize? That’s the Question

            Too many people argue that hypothesis is the ‘if … then’ expectation of the experiment based upon some influential background research and other reasonably strong findings from other studies that have already been done on the same topic. Actually, I would partially agree with that as the argument definitely supports the idea that hypothesis is the premise of the experiment from guesses/estimations which are derived from the knowledge we have got via results of research made earlier. But does this imply that it is always wise and to our benefit to hypothesize before jumping into a research? Seriously, just think about it for a second. What in the earth would you loss if you would do so? Are there any disadvantages of including and following a hypothesis in your study at all? I believe that you already have your answers to these questions ready in your mind. Anyways, I would be honored to share mine with you as follows.

            According to David J. Glass and Ned Hall’s A Brief History of the Hypothesis, the hypothesis is a historic, controversial and complex entity. It clearly has several different philosophical assumptions, ways of understanding its concept and, most of all, applications with their effects and consequences, respectively. The controversy about the hypothesis ranges from it being totally rejected by Sir Isaac Newton to David Hume’s tendency to support it. Hypothesis majorly deals with reasoning as the assumptions and guesses that we would make are based on it. Basically, there are two types of reasoning that are out for grabs in the debates about hypothesis and they are known as deductive reasoning and inductive reasoning. Deductive reasoning is the ‘shrinkage’ of a general case down to a specific instance while inductive reasoning, or induction, is the ‘expansion’ of a specific case(s) to a general rule. So, hypothesis mainly corresponds to a deductive reasoning. It is least-likely that an individual would not read about the very minute details of DNA transcription and translation to hypothesize about the very broad concept of an organism’s behavioral changes; instead it would be much more productive and meaningful to study the environment or the genus that includes the species which is even more general than what is being tested in its appearance in the community and the system’s complexity.

            To clarify my stand, I am neither supporting nor opposing against, completely, induction and deduction. I strongly believe that hypothesis is a type of deductive reasoning and this has fairly both pros and cons into a research. As a phenomenon does not hold true to all the cases presented as evidences, hypothesizing about any test might be a total failure to the experiment by guiding the whole procedure wrongly. I would like to argue that it is highly unwise to trust the whole experiment to an assumption which we might not be sure about or misunderstanding it to the point that it could through the whole project off. Although a hypothesis might be correct and reasonable, that does not mean it is the best way of understanding the experiment and learn all the objectives of it. In general, I would argue that hypothesis is not a necessity in a laboratory experiments and looking for other alternatives would be a better path to follow than keeping it there. I barely agree with Newton when it comes down to this point. There is no need to deny the few advantages of hypothesis unlike him but it is also not necessarily the backbone of the experiment as it can definitely get to become in some cases either. The usage and dependence of most of us on hypothesis is the spread of the positives about it in our natural sciences courses throughout the years and decades of our schooling experiences from generations to generations.

In conclusion, hypothesis can do badly by biasing the results of the experiment to any extent by influencing the researcher. There are infinite possibilities of ‘if … then’s’ from which not even one of them is advantageous for us to understand the concept of the study. There is no single doubt that repeated observations, of course, are tremendously important to have a clearer understanding of a concept; but this does not mean that it is an ideal way of learning new things at all. Guessing is not learning the real deal; is it? I hope that I have answered the questions that have been raised above but this is just my perspective; so, what’s yours? 

  (Word count: 755)           

Work Cited

Glass, DJ and N. Hall. A brief history of the hypothesis. Cell. 2008 Aug 8; 134(3):378-81.

Some Important Techniques and Procedures that I have Used

At the very beginning of my research project, I have learned the procedure of the preparation of the cast an agarose gel. In a short and precise way, the procedure is as follows:

-          measure 50 mL of 1x TAE buffer and pour it in an Erlenmeyer flask;

-          measure and add, into the flask, 0.4 g of agarose (i.e. a white, fine powder);

-          mix the solution by gentle swirling which should give a very cloudy/milky solution;   

-          put the solution in a microwave oven for 60-80 seconds to get a clear solution;

-          let the solution cool down for about 10 minutes;

-          add 50 μL of Ethidium bromide solution* into the warm solution;

*Be careful with this- it's a mutagen and suspected carcinogen, so don't let it spill or get on your hands. 

-     pour the whole solution into the plastic gel chamber and place the plastic comb, with sufficient number of teeth to the number of samples we are testing, to create indentations;

-     wait until the gel would solidify within around 15 minutes;

-     take the comb out and move/place the gel on to the gel apparatus**;

**This is made of hard plastic and consists of an electrophoresis chamber with two wells on the side, a removable gel tray, a comb with as many teeth as you have samples/ladders, and a lid connected to power cords. Place the gel tray in the electrophoresis chamber so that the rubber gaskets on the tray engage the sides of the chamber. You may have to wet the gaskets with water so that they slide in. 

-          fill the gel and the whole apparatus with 1x TAE buffer to cover the sample in depth;

-          then carefully pipette a load of sample into each indentation, recording which sample corresponds with which lane;

-          into one lane alongside those containing your samples, load 5 µL of  DNA ladder, which contains DNA fragments, and 1 µL of 10x concentrated gel loading solution;

-          cover the electrophoresis chamber with the plastic cover, and plug in the two power chords so that the black, negative one is next to the indentations that you have loaded the samples into, and the red, positive one is opposite them;

-          start running the gel for 30 minutes which is indicated by bubbles released; and, finally,

-          remove the gel, place it under a UV lamp, and photograph.

To transform DH5α E. coli with the plasmid:

-          heat shock method was used to transform DH5α cells with the plasmid sample for cloning purposes. 

-          100 μL of CaCl₂ competent cells were divided into two tubes, with one serving as a control tube for the transformation. 

-          3 μL of the plasmid was added to one tube. 

-          Then both samples were heat shocked at 42°C for 45 seconds, 400 μL of NB media added, and incubated with shaking at 37°C for one hour. 

-          The samples were placed on pp LB Broth (100 μL /mL) plates with a sterile stick and incubated over night at 37°C. 

-          Colonies of the transformed cells were then grown in the pp LB Broth medium with shaking for 2 nights at 37°C. 

To isolate the plasmid from the DH5α cells:

-          Two tubes were filled with 1.2 mL of the cell culture.

-          They were centrifuged at 12,000g for 30 seconds at 4°C and the supernatant was removed. 

-          They were mixed by inversion several times and stored on ice. 

-          150 μL of a solution was added to each tube. 

-          The samples were vortexed gently for 10 seconds and incubated on ice for 5 minutes.  They were centrifuged again at 12,000g for 5 minutes at 4°C. 

-          400 μL of each supernatant was transferred to a new tube. 

-          2 volumes (800 μL) of room temperature ethanol was added, the samples mixed by vortexing, and allowed to stand at room temperature for two minutes. 

-          The centrifugation was repeated at 12,000g for 5 minutes at 4°C.  The supernatant was removed and the samples were dried using a speed vac. 

-          Both samples were redissolved in 50 μL TE (pH 8) with RNase (20 μL /mL) by briefly vortexing.

-          The two samples were run on an agarose gel and examined under UV light for analysis of the plasmid bands.

To prepare the buffers for DNA extraction:

-          30 mL of P1 buffer = 0.1818 g Tris base + 0.1116 g Na₂EDTA.2H₂O + 24 mL distilled water + 3 mg RNase A.

-          30 mL of QBT buffer = 1.3149 g NaCl + 0.3138 g MOPS + 24 mL distilled water + 4.5 mL pure isopropanol + 0.45 mL Triton x-100 solution + 0.405 distilled water.

-          200 mL of QC buffer = 11.688 g NaCl + 2.092 g MOPS + 160 mL distilled water + 30 mL pure isopropanol.

-          50 mL of QF buffer = 3.6525 g NaCl + 0.303 g Tris base + 40 mL distilled water + 7.5 mL pure isopropanol.

(Word count: 835)