Happy Friday (or should we say, Fri-YAY) from CauseScience!
psgurel– Today I am miniprepping! If you remember last week, I was doing PCR to get a specific DNA construct. After doing PCR, there are several steps before you have nice clean DNA. For the DNA I’m using (plasmid DNA) the final step is to extract your DNA from bacteria. Lucky for us, several companies make “miniprep” kits that make this process super quick and easy. It takes about 30min, and then you have (hopefully) nice, clean DNA!
crestwind24– This is crazy! I am also doing mini preps of DNA this morning!! SAMESIES!! Preparing DNA is a major part of most labs, as made obvious by todays post. I am making DNA that will label synapses in neurons in C. elegans. Once I have the DNA that I want, we will inject it into developing embryos, and then I will have transgenic worms!! Hopefully with glowing synapses!! This will allow me to visualize connections between different neurons.
CauseScience Friday… more like mini prep Friday!!!
crestwind24– Today I am doing a bunch of VERY serious molecular biology (as you can see from my picture)!! I am extracting DNA from C. elegans and genotyping them for a transgene that I hopefully added to their genome. In short, I performed an experiment to add my DNA of interest to the DNA of the worms, and now I need to check for worms where the addition of my DNA of interest worked. Cross your fingers for me! Maybe this serious face will change to a smiling face by the end of the day!!
pgurel– Today I’m preparing grids for cryo EM. I’m troubleshooting some problems (as I mentioned previously, I’m trying to develop a new method), and the first step of preparing grids is to make sure they are extra clean. One step I take is to clean grids using a glow-discharge plasma treatment. This is basically a fancy way to try and remove all impurities or contaminants from the grid surface. For highly sensitive techniques like cryo EM, even very small impurities (smaller than a speck of dust) can ruin the experiment, so it’s incredibly important to make sure grid surfaces are clean! Here I am with the plasma cleaner!
psgurel – I’ve just spent most of my morning in lab meeting, and will spend my afternoon going to a few lectures. Presenting work and getting feedback is a critical part of science. Lab meetings are somewhat more informal sessions where the entire lab can get together and discuss specific details on members’ projects. It’s helpful for all involved in terms of troubleshooting, fine tuning direction of the research project, etc. It’s fun, but can also be somewhat tiring. Here’s my post lab meeting selfie 🙂
crestwind24 – This morning I came into lab very early to make sure I got all of my experiments done before heading to Philadelphia in the afternoon. One of the things I needed to do was run 4 PCR reactions and then image the products of the reactions using gel electrophoresis. Or more simply, I needed to make specific fragments of DNA from the C. elegans genome, and then make sure I made the correct DNA by looking at its size. When I visualized the PCR reactions separated by size on an agarose gel (top panel), I did not see the size DNA I was making with the PCR. Instead I saw a bunch of randomly sized bands and smears (hence my grumpy face when sitting at the gel imager, bottom left). Luckily, since I was in lab so early, I redid the PCR at a different temperature and got nice crisp bands at the exact size I expected (right bottom panel)!!! I love when molecular biology works!!!
crestwind24- I am spending part of my day processing images (micrographs) that I took earlier on our labs confocal microscope. As I mentioned in a previous CauseScience Friday, the confocal allows me to take amazing pictures of neurons and their axons and dendrites. Today I am taking many images taken through the depth of a worm and making 3D animations. This allows you to see the morphology of the axons – or where they are in space. Below is a partial low-res GIF I made of one of my animations – it shows two neurons and their axons in C. elegans!!
psgurel- Part of joining a new lab involves developing a new project. Today, I’m doing some test runs as an initial step in developing a method for imaging different conformations of actin filaments for cryo Eelectron Microscopy. First, I have to coat EM grids in a mechanism that will allow the actin to bind properly, so I’m surveying different ways of coating EM grids. Wish me luck!
crestwind24– Today I am at an all day meeting in NYC, the New York Area Worm Meeting! Basically, the meeting consists of discussions and presentations from labs that study C. elegans in New York City and the surrounding area! Very excited for a nice day of research talks downtown at NYU!! Followed by sushi with my co-workers this evening!!
psgurel– Getting my hands wet with my first round of experiments in a new lab! I will be doing a lot of structural biology, specifically trying to determine high resolution structures of cytoskeletal elements. One technique I’ll be using in the new lab is Cryo Electron Microscopy. This is different from negative stain EM (as mentioned on a previous CauseScience Friday); the method of preparing samples is different and much trickier: samples are flash frozen in liquid ethane (which is liquid at -180C… COLD) so they maintain their natural/native state. This gives us a much better idea of how these proteins ACTUALLY look. Today I am imaging samples that have already prepared the day before!
After a short hiatus from the holidays, we are bringing back CauseScience Friday!
crestwind24- I have a busy day today, mostly scoring phenotypes of aging worms at my dissecting scope. I am also using an awesome fluorescent dissecting scope, which allows me to look at neurons in aging worms as they crawl around!!
psgurel– New year means new beginnings… This week is my first week at a NEW job, a Postdoc position at the NIH. Today, I’m continuing to get settled into the new lab, and I am planning/organizing/researching my future research project. Fun times!
crestwind24- This morning I am working on my lab’s new confocal microscope (middle). Using this fluorescence microscope, I can take high resolution images of neurons in C. elegans, including their amazing axons and dendrites!! To do this, I use worms that express fluorescent markers in distinct neurons. The fluorescent proteins then fill the neuron cell bodies, and can be excited with lasers to emit light!! I then use the confocals photo multiplier tubes to detect the light and create an image! The image on the right shows 2 neurons and their processes (one red and one green)!
psgurel– I have been spending this morning packing and getting ready for the American Society for Cell Biology (ASCB) Annual Meeting in Philadelphia! This is a 5 day long international conference where thousands of scientists, exhibitors, and students gather to discuss science, network, learn about career opportunities, and more. I’m pretty excited because this year I will be giving a small talk on my research, and I will be participating in a panel on science advocacy. It’s my 5th time attending this Annual Meeting, and I’ll be posting and tweeting throughout the conference.
psgurel: I always save my favorite experiments for Fridays! Today, I’m doing an assay to look at kinetics. ATP (Adenosine triphosphate) is typically known as the main “energy” source in cells and is required for several reactions to take place. On a chemical level in these reactions, ATP gets hydrolyzed and you are left with ADP (Adenosine diphosphate) and phosphate as a product. Today, I’m looking at how fast different proteins hydrolyze ATP. To do this, I stop reactions at various time points, and then I add a green dye that labels free phosphate. The dye turns darker shades of green as the amount of phosphate in solution increases. Check out my samples! The darker the green, the more phosphate is present, which means more ATP has been hydrolyzed!
crestwind24: Today I am scoring and imaging the neurons of old worms. This means sitting for a few hours in a dark room at a big microscope and computer. Specifically, I am trying to see what is happening with certain neurons in the worm as they grow old. We know very little about how neurons change in old age, and why some die in normal aging, as well as in many diseases. In the first picture you can see the green light under the microscope, which is activating my fluorescent proteins. On the computer screen you can see the image the microscope is taking. The arrows point to the 2 worms I was taking picture of, old worm selfie!! The selfies are a bit grainy since the room is dark… sorry … and its time to upgrade my phone.
crestwind24: Today I am doing PCR to amplify a gene I am interested in from the DNA of a worm (C. elegans). PCR, or Polymerase Chain Reaction, can be used to make DNA, check for the presence of DNA, and/or sequence DNA. PCR is commonly used to detect the presence of viruses, like Ebola, by looking for the DNA of the virus in the patient’s blood or bodily fluids. Setting up a PCR involves pipetting small volumes of liquid, containing DNA, enzymes, salts, etc into tiny tubes (see images). Then the tubes are placed into a cycling machine (ours is named Cycle Jackson… get it?) that changes temperature over and over in a cycle to activate the enzyme that makes the DNA. At the end I hopefully will have a bunch of the DNA I want!
psgurel: Today I am staining grids for Negative StainElectron Microscopy. I will be using the TEM (Transmission Electron Microscope) which essentially uses a high voltage electron beam to visualize samples. My samples are placed on tiny grids (the arrow in my picture) and then stained with uranyl acetate, which is slightly radioactive (hence why I have to wear a lab coat) and scatters the electron beam. As a result, my samples stained with uranyl acetate will not absorb electrons and thus I can visualize them in contrast to the grid surface which will absorb electrons. Why use TEM instead of other types of microscopy? A typical fluorescence microscope yields about 200nm resolution. However, I’m trying to visualize protein clusters of less than 100nm in length…about 10,000x smaller than a grain of sand! The TEM will be able to resolve these structures!