protein

Co-IPs are a highly specialized approach to identifying in vivo protein:protein interactions. The protein preparation is fairly similar to a total protein prep, however, a different buffer is used for lysis, and a column in used to run the fraction through. Our lab uses micro-max columns which consist of thousands individual iron atoms fused to a primary antibody (typically C-myc or HA). Running the intial fraction through this column will grab the input, or protein complex that you are initially probing, in other words, one of the two anticipated interactors. This fraction is quantified/normalized to other samples that are later run out via SDS-PAGE. This is then transferred to a charged membrane that can be probed with a different specific antibodies, which would detect the secondary interacting protein. It is crucial to rinse or wash the membrane between probing steps to eliminate any risidual or background primary antibody that has unspecifically bound to the membrane. 

protein

Protein protein interactions in vivo are a tough phenomenon to capture. However, there are special ways to identify proteins that are capable of interacting in vivo. One approach is a method called co-immunoprecipitation (Co-IP). This is an extension off of the simplistic approach of western blotting, or, immunoblotting. This technique takes advantage of biology at its core. Scientist have developed a method to engineer antibodies that are capable to bind to whatever target, or antigen that is desired. The process begins by injecting the target protein into a host (rabbit, goat, donkey, etc…), which then generates antibodies that bind to the “foreign” protein. The animal is then bled, and syrum is isolated, and the specific protein (primary antibody) is purified. The secondary antibody is synthesized in a specific manner, where it targets the “species-specific” portion of the primary antibody (anti-rat, goat secondary). Once the appropriate / specific antibodies have been made, immunoblotting can ensue. 

round II

Before dinner, the family drank a couple of beers, and this alone is enough to stir inspiration up in this family. Shortly there after, we decided to go to the community recreational center to lay a couple of games of basketball. I was not prepared. I borrowed a pair of gym shorts from my cousin (which was okay), but shoes were my biggest issue. I had to wear semi-formal dress shoes to the gym. Can you see the potential problem at hand? We are a fairly competitive family, naturally, so I played hard throughout the first three games. At the end of this game, I decided that my feet were in trouble. I sat down and checked out my boats. Sure enough, I could tell I had developed blisters during the second game, what I did not know was the severity of the blisters. My socks were blood stained, as well as the soles of my shoes. Despite this set back, Thanksgiving was awesome.

round I

Last weekend I had Thanksgiving weekend in the Dallas area with my Dad’s side of the family. It is a serious advantage having Thanksgiving practice one week before the real deal. This allows for some stomach-stretching to occur. This is analogous to practicing for any sporting event. This time we had two turkeys, one was smoked by a BBQ restaurant in the Dallas area, Dickie’s, and the other was smoked at my uncle’s house by the man himself. It is a toss up as to which bird I preffered, but since I’m an original kind of guy, I’m obviously going to pick the turkey that was prepared in my uncle’s house. Honestly, both were equally fulfilling. The dressings were something out of this world. Both were pan-baked dressings, thick in nature, thick enough to stay attached to an inverted spoon. Cranberry sauce was NOT out of the can. The sweet aroma of the simmering cranberries filled the house. 

bakn a chkn

After my hour and 15 minute timer expires, I remove the lid and bake for another half hour or so (depending on the size of the chicken, of course). This secondary baking strategy allows the skin to crisp up a little bit and allows the internal temperature of the chicken to reach the appropriate temperature of 165 degrees. I allow the chicken to cool without a lid for about half an hour before I start carving. After all of the meat has been removed from the carcass, I will often times boil the skeletal remains to produce hearty chicken broth that I will use at a later date. In the case of last week, I make about 10 cups of chicken broth, froze 5 cups, and proceeded to use 5 cups for the base of my chicken soup, a culinary zeal rushed over me, there is not much else I can say.

bakn a chkn

I had myself a nice, relaxing weekend. Mostly this occurred as an absence of OU fans invading my town. Sunday was especially relaxing, as this was the day when I baked a whole chicken. In order to bake a chicken properly, there are a couple of ground rules that need to be followed. First, you must clean the chicken. I typically do this under cold water. During this “cleaning” process, I will take the giblets out of the body cavity, and set them aside. Next, I will pat down the outside of the chicken with paper towels. After the skin has dried a bit, I drizzle a fair amount of extra virgin olive oil on the skin and spread it around with my hands. I then rub kosher salt into the skin of the chicken. After these preparation steps have been taken, it is time to start baking. I cover the casserole dish with a glass lid and bake at 350 degrees for about an hour and 15 minutes.

okay

I just made a serious noob move in the lab, fortunately it was for the best and was overlooked as the mistake that I thought it was. Here is how it went down. One of our mutants lines in the lab is resistant to a herbicide called BASTA. Most of the plant expression vectors that I use are also BASTA resistant. Is the mistake clear yet? I cloned my insert into this plant expression vector with the identical resistant marker as the plant that I was planning on dropping the clone into. The problem is that I would never be able to select for positive transformant if both resistance markers are the same. When spraying with BASTA, I would only get positive mutant plants back, I would not be informaed that I got a mutant plant with a positively inserted transgene. This expression clone is fine for selection of transgenic plant in wild-type plants, which I negligibly forgot we were doing.. 

eating

As of late, I have been eating very poorly. Not that I voluntarily choose to eat bad, I have just been extremely busy, and the last thing I want to do when I get home is cook. On the menu for the last week or two has been a poor selection of pizza shuttle, Whataburger, Subway, and most importantly (but definitely night the healthiest choice), Pepe’s. Starting this week I have made a personal agreement not to eat out. Lunch today, however, was fantastic. I had steamed rice with fresh onions, broccoli, soy sauce, siracha hot sauce, salt, and pepper. A spinach salad with poppy seed dressing and dried cranberries complemented my rice dish quite well. The only thing that should/could be improved is topping my salad with a nice ripe half of an avocado. This fruit meshes perfectly with the tasty matrix of the spinach salad. Eating well makes me feel healthy, which is a necessity when putting in long days. 

sammy

How about the day after Thanksgiving? I’ll tell you about that day, and the following week or two. It’s Thanksgiving sandwich week. If you are not familiar with the concept of the “Thanksgiving sandwich”, get ready. I would suggest making this sandwich on generously sliced bread, ignoring this mundane detail will most definitely result in a messy explosion. Lay one piece of bread down, spread fresh cranberry sauce over this slice. Next, lay down a thick bed of turkey (dark or white meat will do). Turkey is a serious deal in my family. My uncle always prepares the bird by pureeing half a gallon of fresh jalapenos. This puree is then injected via syringe into the bird as if being shot by a pepper shotgun. Cover turkey layer with dressing, preferably not the crumbly grumblies at the bottom of the Tupperware or ziplock bag, I’m talking about the fresh, moist goodness. Cover this layer with another piece of bread with mayo, and enjoy.

thanks

Thanksgiving is a time for being thankful for everything that the fortunate are granted, whether be it a result of hard work, or simply luck. It is also a time to eat. I’m not trying to take away from the importance of appreciation of everything we are given, nor am I saying that we shouldn’t give. All I am trying to stress is that we should seriously think about this holiday coming up. Every year, I neglect Thanksgiving until I finally wake up and it is Thanksgiving day, and before I know it, I am already knee deep in mashed potatoes. After eating, I find myself watching a Dallas Cowboys game, that I rarely ever have interest in, independent of who they are playing. I am suggesting that we implement some sort of a countdown, similar to Christmas. We need to get excited about this happening, and never let this glorious day slip away ever again. 

honey bbq chicken strip sandwich

Apparently the McRib is back. Do you think McDonalds temporally ties the revisiting of the mcrib to the monopoly game that they play? They call it a game but it has probably taken more than a couple of lives. These terrible companies are amazing at marketing if this was intended. I view the introduction of the mcrib analogous to whataburger’s long-awaited, monthly special that occurs only once per year: the honey bbq chicken strip sandwich. This special is not even that good to be honest, its just the constraint on its availability that keeps me from being able to resist. I showed up a day after they switched the monthly special from the honey bbq chicken strip sandwich to some other bullshit, and they refused to serve me what I came for. I asked them, “do you guys have chicken strips?” They replied, “Yea”. I then proceeded to ask, “Do you have buns and cheese?” I got the same reply. Then, I was nervous when I asked this question, since it is inherent that the sandwich would be worthless without this element, “.. and you guys have bbq sauce?” I got the same reply. I then asked if they could “make me a sandwich with the following ingredients.” THEY SAID NO.

gutter

As of late, I have been listening to an unreal amount of Lil Wayne. Even when writing, I cannot seem to escape him. I have come to the conclusion that it is not the lyrics that I prefer, but rather, the beat. If it were the lyrics I was after, I would hypothesize that I would have a difficult efficiently writing while listening to the music. Most of the beats he produces seem to carry a natural vibratory oscillation, which makes for easy listening. The beats flow and seem to remain at a high enough beats per minute to avoid putting me to sleep after long workdays. Maybe it’s not such a Lil Wayne fixation as it is a rap fixation in general. Wiz Khalifa is also high on my list right now. Only a few tracks from his albums have I added to a listenable list. I find the majority of his album tracks to be boring and mainstream. Wiz mixtapes on the other hand are quite the opposite. I am guessing that he doesn’t have to be the role model in this venue as he does for platinum record venues. Likewise, the beats produced by Wiz promote writing and keep me in an update mood.

selecting the parent

One note on picking which genotype should be represented by the male and female, respectively, should depend on the selectable marker of the male, or in other words, whatever selectable marker is associated with the gene being crossed into the female background. This acts as the experiment and as an internal positive control for the cross itself. For example, if the female is a mutant line that does not harbor a resistance marker, it would be advantageous to use a male that has a robust resistance marker associated with its transgene. A couple of examples of robust molecular plant markers would be BASTA (a herbicidal resistance gene), Kanamycin, or hygromycin. If the male parent is homozygous for the BASTA resistance marker, one can harbor seeds from the cross, plant F1s, and spray these plants with BASTA. Whatever lives should represent a productive cross that is heterozygous for the male transgene. 

crosses

Today I set up my first crosses in Arabidopsis thaliana. This is going to be a brief explanation of how to make crosses, but should provide enough information for anyone to perform an efficient cross between two parental lines. The first step is selecting an appropriately aged female. The proper age should be one primary bolt with no more than 3-4 flowers opened. Arabidopsis is an organism that self- pollinates, so one should be extremely careful to select flowers to emasculate that have not previously been pollinated. Emasculation should be performed with extreme caution. The goal here is to remove all male reproductive organs, namely, the anthers. After all anthers, sepals, and petals have been removed, the stigma, or female reproductive organ should stand-alone. After the flower has been emasculated, the plant should be set-aside for one day, until a crown has formed at the tip of the stigma. This morphological distinction represents a receptive stigma. This is the time that artificial pollination should take place. One should repeat pollination once a day for three consecutive days after the stigma has become receptive. 

rage

So last week my computer crashed, and of course, I had to give a talk three days after the catastrophe. Luckily, I was able to use the boss’ computer to give my seminar presentation to a new journal club our lab recently joined. I washed away the nerves and gave the talk. I feel the overall flow of the presentation was quite fluid (I did get a chance to practice this talk at another venue). Maybe this little tidbit of information made this talk a little easier to give. As I am trying to blog about this senselessness, I am being distracted by one of my lab mates, she’s quite a unique case (I should inform you of this so you disbelieve any of the following). She is frantically trying to finish her homework assignment for a class that we are late to right now. In her attempt to get this assignment completed, she is ranting and raving in a belligerent manner. CUSSING. 

Things on my desk IV

I also have a half drinkin cup of Starbuck’s Italian Roast coffee, a half eaten lemon cake (also a Starbuck’s purchase), sunglasses, phone, another used paper towel, a green nalgene water bottle (half full of reverse osmosis treated water, pH 6.5), and finally a very special black pen. I find this pen to be special and confusing, synergistically. On the side of the slender pen, reads “Le Pen”. This would lead you to believe that it is a French item. If you were to make this assumption, you would be sorely mistaken. The pen was actually made in Japan. Do you see the confusion? I think that is all. Actually, I am positive that is all. Nope, I left something out. A scotch tape dispenser with approximately 2/3 of a full role of translucent tape. This is good for taping things. It seems as if I have just received a text message.

Things on my desk III

On the right side of my desk: I have a white three-ringed folder with the instructions of “how to teach Dr. Holts Principles of Plant Physiology handbook”. I don’t know why I put that it quotes; it has been a rather useful resource for me this semester, as this is the first semester I have taught the lab section of this course. Atop my handbook, are some random papers that I don’t care to thumb through, as I don’t think it will affect the outcome of your day? Atop the stack of white papers, is my lab notebook. This is a sensitive subject, so this is another portion of my desk collection that I am choosing not to elaborate on. Due east of my lab notebook, useless white papers [I don’t know if they are useless (they probably contain some data that I collected and never bothered to jot down in my lab notebook)], and handbook stack lies my wallet. I have a ten-dollar bill and two one-dollar bills inside of it.

Things on my desk II

In addition to what I have already told you, I have a weathered green post-it note that has the following words written on it (in no specific order): Dad, OG+E, city, autoclave (with a smiley face?) Sony cybershot 8.1 megapixels super steady shot dsc t100, and finally (this is the best part) “cat mother and the all night news boys”. I think this might be a band that I was supposed to look into, however, I cannot back that statement up to any regard, so forget about it. I have my bio-writing textbook that is marked with a Korean bookmark on chapter 17. I have 11 coins on my desk. Of these 11 coins, 3 are quarters, 6 are nickels, 1 penny, and 1 dime – this totals to $1.16. For what its worth, I have no idea what you can get for that amount of chump change anymore. There is an iPod connection cord also. 

Things on my desk I

At this current moment I have on my desk: one blue and grey backpack that has no less than seven zippers. I have a Tupperware container that never seems to get as clean as it was it the time I purchased it, actually, it always looks dirty, I’m not trying to make this sound like something it isn’t. I have a pair of elementary ruled scissors with red handles. These scissors seem to be sharp enough to chop through an index finger bone. I have two full-sized legal pads and one carry-size legal pad, exclusively for “on the fly” note taking. I have a couple of used napkins, regrettably enough. There rests a pair of studio Sony headphones that I use to avoid talking to my lab mates. I have several scientific papers spanning the highly acclaimed scientific journals of Science, The Plant Cell, Plant physiology, Nature, and PNAS. Underneath my desktop computer, I have small Tupperware containing 3-month-old craisens. 

undergrads3

I am hoping that she will catch on to genomic DNA preps and PCR rather quickly. Because I have an upcoming project that is going to require an extreme amount of both of these techniques. After she feels comfortable with genotyping plants, we will move on to more advanced tasks such as protein analysis, gene expression analysis, and eventually molecular cloning.  I have witnessed and been told that it takes a year or more for an individual to become competent in a molecular biology lab – that is, they do not need someone to be watching their every move, they can undertake real responsibilities pertaining to the research being performed in the lab, and most importantly, obtain the ability to think independently at the level required to work in a cutting-edge molecular biology lab. I have high hopes for this undergrad, as she attended one of the most prestigious science high schools in Oklahoma. All of my explanations seem to be sinking in with ease, however, I shouldn’t speak so soon – we will see how this PCR looks…

undergrads 2

We are performing PCR to identify if 35S:NF-YB12:YFP:HA was ever inserter into the genome. We will be amplifying the region using a 35S forward primer and a gene specific reverse primer. This should tell us if the transgene made it into the genome. We could also amplify another region, using a YFP reverse and gene specific forward primer combination. We could amplify another region, using the primer combination of gene specific forward and HA reverse. One final combination, which would give you the answer, but would be a sloppy approach is using the two primer extremities of the construct, 35S forward and HA reverse. This requires doing a little math for confirmation. This requires looking at the sequence of the construct, counting nucleotides for each portion of the construct, and adding this together to get a collective base count of the construct. The amplified band should be exactly that size.

undergrads

I am currently working with a freshman undergraduate student who wants to graduate with a degree in pharmacy. Hopefully exposure to a molecular biology lab will steer her in the correct direction. Yesterday I walked her through the workings of a plant genomic DNA prep. It looks like the DNA we extracted is going to work. We will find out the answer today. We are testing whether or not the prep worked by PCR. She will be amplifying a region of DNA where I suspect there to be a transgene. I am asking her to help me with identifying the presence of 35S:NF-YB12:YFP:HA. I am a bit skeptical of this line because I have performed protein analysis and had odd results. Most of the proteins I am trying to detect are ~25 kDa, with a 27 kDa YFP marker. This should some to ~50 kDa. I am worried because I am getting bands on western blots that are only half that size. This tells me one of two things: either the protein of interest is being cleaved out, and I am detecting only YFP:HA, or the other scenario, which is that I have no idea. 

the dark side of translational fusions

Inhibitory effects of protein function are usually associated with mutations within the coding region of a gene. Inhibition can also come from additions of elements to a protein. I was reading a paper recently that showed that the C-terminal addition of GREEN FLUORESCENT PROTEIN (GFP) to a gene construction actually affects the ability of the protein to perform, in vivo. In order for plants to flower, a small protein (~25 kDa), called FLOWERING LOCUS T (FT) must be transported out of the leaves, through the petiole, loaded into the phloem, up through the plants vasculature, where it eventually reaches the shoot apical meristem to induce flowering. The small nature of this protein, accompanied with the long distance it must travel to accomplish one of its jobs, can be negatively affected by the addition of GFP, used to detect sub-cellular localization.  The translational fusion of GFP to FT decreases the efficiency of FT to drive flowering in an ft mutant background. This is supported by flowering time assays in Arabidopsis.

PAGE

Today I looked at protein expression of a flowering time experiment. I evaluated protein expression by western blotting. This is a fairly straight-forward concept that allows you to see relative amounts of protein. I began by extracting total protein from plants that I was interested in evaluating protein expression/accumulation. This process starts by clipping a single leaf from a plant and grinding the leaf in a tube with an appropriate amount of extraction buffer. The extraction buffer allows the protein to go into solution with a cold centrifugation step. After centrifugation, everything except total protein is pelleted to the bottom of the tube. I then remove this top portion, called the supernatant. This protein rich extract is transferred to a tube containing loading dye, and heated at 95 degrees for 5 minutes. This heating step denatures the protein, or linearizes it, in other words. We are now ready to load our proteins into a poly-acrylamide gel that utilizes charge to push the proteins through the gel. The different proteins are separated by size, larger proteins remain higher up on the gel, smaller ones drift further down.

Watson and Crack, fleshed out.

We wish to suggest a structure for the salt of deoxyribose nucleic acid (DNA). This structure has novel features that are of considerable biological interest in that it suggests a copying mechanism for genetic material.

            Several structures for DNA have been suggested, but all are unsatisfactory in our opinion. Our structure uses molecular architecture similar to Fraser’s model, proposing that phosphates occupy the outer extremities of the chain, with the bases making up the central region. We propose that DNA consist of two helical chains each coiled around a central axis. Both chains follow right-handed helices, however, their orientations are anti-parallel. Each chain is composed of a phosphate-deoxyribose backbone, with 3’-5’ linkages.

            Four molecules make up the structure of DNA, two pyprimidines, cytosine and thymine; and two purines, adenine and guanine. For bonding to occur, specific purines must join by hydrogen bonding with specific pyrimidines: adenine to thymine and cytosine to guanine, respectively. Bonding specificity is consequence of the molecules taking keto-configuaration. Our model is experimentally supported by reports claiming that ratios of adenine to thymine, and cytosine and guanine are always close to unity in DNA.

            This model is biologically significant because it proposes that phosphates are readily available to be accessed by the cell’s excess of cations. Our model also assumes that DNA is an open, hydrophilic molecule, thus, highly water-soluble. Alternatively, at lower water content, DNA’s thermodynamic favorability is predicted to tilt its bases so that the structure can become more compact.

            

flowering

Light has a strong effect on the stability of proteins that regulate many physiological and developmental processes in animals and plants alike. Circadian rhythms depend on rapid accumulation and degradation of protein products within a 24-hour time window. Light to plants is more than a source of energy to generate sugars; it is an external timing apparatus (for most plants) that allows for the plant to reproduce when environmental conditions are favorable for offspring development. For floral initiation in Arabidopsis to take place, 16 hours of light per 24-hour period, is preferred. Light strikes the photoreceptors, PHYA, PHYB, and CRY2, which then entrain the circadian clock to trigger the expression of CONSTANS (CO). CO is a transcriptional regulator that is expressed (under appropriate conditions) in the leaf vasculature in most long day (16 hours of light, 8 hours of darkness).  CO expression follows an oscillatory trend throughout a 24-hour period, as the protein accumulates in light and is rapidly degraded in the dark. With the help of the transcriptional regulators that the Holt Lab studies (NF-Y), CO can bind to the promoter of a gene called FLOWERING LOCUS-T (FT). FT acts as the central hub for many flowering pathways. After FT has been activated, the protein product (produced in the leaves) travels against a natural gradient, through the phloem companion cells, where it eventually reaches the shoot apical meristem to turn on another group of transcription factors that induce flowering. 

flowering

Plants, like animals, respond to environmental factors. Light seems to be the most influential external factor on the planet (omitting the anomaly of hydrothermal vent life of oceanic trenches). Light is key to plant life for synthesis of usable sugars, as well as reproduction. Floral initiation, in Arabidopsis can occur through a number of pathways, given that Arabidopsis is a facultative, long day plant. This means that is favorable for Arabidopsis to flower when day lengths stretch into 16 hours of light and 8 hours of darkness, per 24-hour period. Alternative floral initiation pathways can be triggered if this light regiment is not met. An alternate pathway group is influenced by temperature acting as the environmental factor. Vernalization, autonomous, and ambient floral initiation pathways are triggered by variable temperature exposure. A pathway independent of environmental factors would be the Gibberellic Acid pathway. This is a plant hormone that is known to positively regulate seed germination, floral initiation, and many other physiological and developmental processes. 

RNA IV

Poly-A mRNA purification starts by linearizing your total RNA. This is a necessary step because it is thermodynamically favorable for RNA to hybridize back onto itself, creating secondary structures called “hairpin loops”. We must linearize our RNA to remove secondary structures, which ultimately exposes the polyadenylated tail of the mRNA transcripts. After this step, we add our linearized transcripts into a magnetic column containing single thymine molecules (adenines bind to thymines) bound to single Iron atoms. Through binding affinity, we are able to grab individual mRNA transcripts, while allowing any molecules lacking polyadentylated tails to flow through the column. The mRNAs are stuck to the wall of the tube, bound by the iron atom/thymine matrix, which in turn is magnetically attracted to the wall of the tube that is placed in a slot of a device possessing a strong charge, opposite that of iron. We are now ready to elute our purified mRNA. I choose to do this with RNAse free water. I once again determine quality and quantity of my extract by spectrophotometry. 

RNA III

After total RNA isolation and a successful DNA digestion, we need to purify mRNA from our total RNA extract. Total RNA consist of somewhere between 1-5% mRNA (organism dependent) and the other ~95% contains ribosomal RNAs, TRNAs and other small RNAs. Eukaryotic systems have an advantage over prokaryotic systems due to a specific biochemical advantage at the 3’ end of each mRNA transcript. This advantage is a polyadenylation event, or a 15-30 base pair adenine rich tail (consistent adenine repeat) at the end of each transcript. This is advantageous because we can design a “handle” that will, without fail, hybridize to our polyadenylated transcript. In order to isolate an efficient amount of mRNA for RNA-seq (~500ng) you need to start with a large amount of total RNA (remember that only 5% of total RNA are mRNAs), something around 3mg of total should be efficient. Quality of mRNA input into an RNA-seq experiment is extremely important. If care is not taken in the cleanup step, you might as well start over.

RNA II

We start the protocol by freezing our samples immediately in liquid nitrogen to solidify the transcript that we are going to capture, this also disrupt any RNAse activity that could potentially degrade our transcript. We then grind our frozen tissue a in a frozen mortar with an RNA extraction/lysis buffer. We add this slurry of tissue/buffer into a homogenization column and centrifuge to filter through the RNA rich liquid we are after. Next comes the precipitation of RNA. This is facilitated by absolute ethanol. We add the ethanol/buffer+RNA mixture to a separate column that has high binding affinity to RNA. The RNA precipitates after a short spin and binds to the column matrix. We then add several wash buffers to “clean up” our sample that is soon to be eluted.  The second wash buffer contains ethanol that has been diluted to 70%. This allows for both ethanol and water soluble contaminants to be discarded. While isolating RNA, there is always genomic DNA contamination. This occurs due to shared biochemical properties between the two molecules. I prefer to do an “on column” RNAse free DNAse treatment that supposedly degrades 99.99% of contaminating genomic DNA. After this step, we are ready to elute our clean, total RNA. I use a spectrophotometer to determine the quality and quantity of my extraction.

RNA I

The overarching goal of my Transcriptomics (RNA sequencing) class is to provide an outline, or protocol to send samples for RNA-seq that would work for anyone working in our system. This is a great idea because we are getting a wide range of protocols covering a wide range of systems. All eukaryotic systems are virtually identical, however, the prokaryotic crowd encompasses aerobic and anaerobic organisms with radically variable needs.

The starting place for an RNA-seq protocol begins with total RNA isolation. This process differs between plants and animals due to the presence of a cell wall in plants. Plant RNA isolation is a fairly simple process, if using a kit. Before one can begin to isolate this fragile nucleic acid, certain procedures must be taken and potential problems must be addressed. First, RNA is single stranded, thus lacking the hydrogen-bonding matrix that DNA, a double stranded nucleic acid contains. This property makes RNA very fragile. Second, our skin, breath, and hair are riddled with proteins called RNASes. These are proteins that target RNA and chop the transcripts into untranscribable messages, thus destroying the polynucleotide. While isolating RNA, one must not take these two considerations lightly. We ALWAYS use gloves and RNAse/DNAse free materials including water, tubes, isolation columns, and all solutions used throughout the isolation process, as well as any downstream single stranded nucleic acid application.

Natural Variation

Evolution is a driving process that takes in information as an input (typically genetically inheritable information) and spits out deviants or variants of this genetic template. Natural variation among species is the results of continual evolution. Natural variation occurs at all hierarchical levels from kingdoms to families to genus to species. For example, the organism used as a model species in the plant biology world is Arabidopsis. Since the early 21^st century the Arabidopsis community has undertaken the laborious task known as the one thousand and one genome project. In other words, they are sequencing the genome of every Arabidopsis ecotype. We use three of these fully sequenced ecotypes in our lab for various reasons – simply, some are better for certain assays than others. This is an example of natural variation at a large, topical level. Let us consider natural variation at the molecular level. Just as whole genomes differ slightly from their common ancestor, the family of proteins I work on has a large region of homology (~70%) with one another. The landscape at the sequence level, protein and DNA alike follow a trend of each other. It is unknown whether or not these variations account for evolutionarily induced new functions of the proteins themselves. However, the answers are being slowly uncovered. 

untitled

Scientific writing can go one of two ways, interpretable or the opposite. I feel the “middle ground” in scientific writing is under or less represented than writing of other backgrounds – you either get the message or you don’t. It is difficult for a research scientist to step out of a world filled with acronyms and lab jargon to get a relatively (in most cases) simple idea across to the general public. This might be the most challenging task that scientist face today. Whether it is information the researcher is trying to convey through a talk or presentation, write-up or report of some fashion, we are challenged by the task of relaying information. It is possible that the ideas and information come so clearly to researchers in this field because we live in a world that is riddled with simple and complex variants of the central dogma. The same could be said for any professional that finds his or her work inherently and naturally understandable because they are engulfed in the world. Relaying complex information’s requires one to step back, and ask what is the take home message here, and how can I get to that point as clearly and concisely as possible? I have learned that making analogies to my research to be one of the more effective methods of describing my research to people outside of the scientific community. 

Boulder, CO cont.

Next, we went to Pearl street, this is the awesome street with the wide array of food choices, awesome little shops, street musicians, basically the street that every perfect college town has laying behind its campus. Massachusetts street in Lawrence, Kansas and State Street in Madison, Wisconsin are examples. There was a cool restaurant tucked away in a pedestrian zone about half a mile into our walk. We drank our beers on the rooftop patio overlooking the mountains. We spent four hours I'm boulder and in that short amount of time I was convinced that it is bar far one of my favorite college towns. The weather was a beautiful 70 degrees and sunny, I guess they experience an earlier exposure to fall. Driving back into Oklahoma, I had noticed the temperature was roughly identical to what I had become used to while spending a weekend in CO. This surprised and excited me at the same time – I had left OK when temperatures had remained constant for months above 100 degrees. There is hope on the horizon.  

Boulder, CO

The drive into boulder from Denver was about 30 minute trek into canyon surrounded by towering peaks. We had initially planned to meet up with one of our friends from Norman that recently moved to boulder to peruse a PhD in chemistry. After meeting our friend Jesse, We hopped in the car and headed to the CU campus. I had heard people criticize Colorado for not giving the best college degrees, and that it's only a party school. I can see how this might be true, there is just so much to do, and every single day the weather is perfect- quite a change from what I am used to here in Norman. Everything was lush green, alive, and not 900 degrees. Just a glance to the left on a hill and enormous peaks reveal themselves. We walked from the library to the chemistry building, passing over two streams, and a ten-foot sundial in the process.