Reading post!

I was given this reading over Thanksgiving break, so I'm not quite sure what to put as the date (sorry Mr. Calos!)
Anyway, when I went to NYSM over Thanksgiving break, Dr. Kirchman gave me an article that he and some of his peers had written about the North American ivory-billed woodpeckers.
I'm not quite sure how to get the reading on here, but here is a link: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1686174/
Though I admit that some of  the vocabulary went over my head, I found the article to be interesting as well as informative overall. Basically Dr. Kirchman and his colleagues used mitochondrial DNA to compare the ivory-billed woodpeckers and Cuban woodpeckers which are very similar to the ivory-billed.  When Dr. Kirchman decoded the DNA, however, it was found that the two species of woodpecker had split off in the Mid-Pleistocene (more than one million years ago). The North American ivory-billed woodpecker was thought to be extinct until there were reported sightings of the bird in 2005, and by decoding the DNA in this experiment, Dr. Kirchman hoped to create a foundation that could test whether the sighted bird was really an ivory-billed woodpecker by comparing the DNA of a dropped feather or feces sample to his DNA sequence. There were no conclusive conformations of the ivory-billed woodpecker sighting, though, and no feces samples or feather were ever found to confirm a positive match.

<-- woodpecker :)

Gels and pipet tips and sunburns (oh my!)

2/6/12
Today I had a lot of fun in the lab. Dr. Kirchman started off by telling me about another PCR he had run. In order to sequence the DNA, we are sending only the section of the DNA that we need to be sequenced rather than the whole thing. However, we need to make sure that this section of DNA is in good enough quality to be sequenced. Dr. Kirchman tested some of the PCR product and while there was some DNA there, it was very faint and not a bright band of good quality DNA. Today, we ran a larger amount of the PCR product (20 microliters) through a gel in order to hopefully get better quality DNA. This was no ordinary gel though. This was a gel that my own two hands created without Dr. Kirchman even in the lab with me :D I even avoided microwave explosions and air bubbles in my gel, and was deemed worthy of a thumbs up by Dr. Kirchman thanks to my nice work. While Dr. Kirchman quickly loaded some DNA samples from our latest three PCRs (choosing the samples with the highest quality DNA), I restocked the boxes that held pipet tips, improving my hand eye coordination in the process (I've noticed a trend where everything that has to do with DNA is usually tiny).
Then came the really fun part. After running the DNA through my gel, Dr. Kirchman and I used the big machine that produced ultraviolet light (that's where I got the pictures of the previous PCR's a couple posts below) to cut out the DNA from the gel. While I didn't quite have the hand eye coordination to participate (as I told Dr. Kirchman, I was never good at the game Operation as a kid) I donned a pair of protective goggles and stared captivated at the glowing strips of DNA along with how easy Dr. Kirchman made it seem to cut out minuscule blocks of DNA. Dr. Kirchman had to be very careful while cutting out the DNA, however, and make sure his sleeves and gloves were covering his skin, because it is possible for the UV rays to give you a sunburn.
After cutting out the DNA strips, Dr. Kirchman and I used a kit to clean up the DNA, or at least started to use the kit until we ran out of time. We first had to incubate the blocks of DNA and a mixture from the kit in order to melt down the gel.  The blocks of DNA were put in test tubes with 150 microliters of the mixture and then put in a little boat-type tray that would float in a machine that would keep the water at 50 degrees celcius. Special tubes that come with the kit contain filters that contain things that will cause the DNA to bind  to them, but allow the other stuff to filter through. Hopefully, when we are done with the kit, we will be left with some good quality DNA ready to be sequenced.

Long weekend!

1/30/12
Today was a long weekend and I did not go to NYSM :(

AHHH overwhelmingness in the lab :)

1/23/12
I still keep forgetting that it is 2012 :) Anyway, there were luckily no transportation mishaps for me today and the shuttle ride to and from the internship went smoothly (hurrah!). When I got to NYSM, Dr. Kirchman and I quickly jumped into the lab and I got to make another gel for another gel for a PCR. Sadly, me being a newbie, something had to go wrong. I'm sorry I can never remember the names of anything, but the powder that mix with water which you heat up in the microwave that cools (after you throw in a mutagen) and forms the gel... well there was a microwave incident. When you heat up the powder water mixture in the microwave to get it to dissolve, it gets VERY bubbly VERY quickly. I was struggling trying to see through the microwave door and well, I allowed a little volcano to form in the microwave. After cleaning up the bubbly over spill Dr. Kirchman had me pop in a strip or two of some gel from old PCRs into the flask containing what was left of our water powder mixture. You can melt down strips like these and make an entire recycled gel if you have enough, but we just melted a couple in what was left of our mixture to create what was fairly close to the original amount. After putting a little too much of the buffer (the buffer is just water, but the really clean sciency water :D) in the gel holding device (I really couldn't tell you... i think the brand is called owl maybe?) I poured my melted water powder mixture into the container and put in a comb to make the wells in which we would put the DNA.
Since we had to wait for the gel to set, Dr. Kirchman took me to the range (where they keep all the skeletons and stuffed animals) to help him find some specimens he would be showing to a class later that day to illustrate the evolution of limbs. For example, Dr. Kirchman's friend, Joe, who also works at NYSM showed us a mole humerus. The mole's arms had adapted so that they were perfect for shoveling dirt and the humerus was at an angle so it would be easier for the mole to scoop (http://www.mnh.si.edu/mna/full_image.cfm?image_id=834 <-- mole arm :D).
After freeing an emu from a crate (with the help of a screwdriver, scissors, and wire cutters), we went back to the lab to see that our gel had turned opaque and was ready for us to place the DNA in the wells. Putting the DNA in the wells, however, takes quite a steady hand. My hands are not that steady. However, I was doing pretty well until Dr. Kirchman commented on how well I was doing. With my chest puffed out as I began to put the 7th sample of DNA in a well..... I pushed the pipet down into the bottom of the well too hard which meant the DNA couldn't come out of the pipet and when i removed the pipet the DNA splooshed everywhere but inside the gel. Whoops. Dr. Kirchman quickly whipped up another sample of that same DNA, fixed my mistake, and I finished the last two wells successfully. Overall it was a good day in the lab, mistakes and all, because practice makes perfect :). Hopefully it will just be a little more perfect next time.

PCR pictures :D

This is PCR 169 and it is the first PCR where we were actually able to see some nice rail DNA without the scary wiggly streaky things (possibly caused by the snow-flaky substance in the DNA). After Dr. Kirchman cleaned up the DNA removing any proteins our other materials that could have contaminated it during extraction, we were able to see the DNA even though it wasn't very pretty. The first test tube with the nicer looking DNA is a control that contains a DNA sample that we know will work to make sure that if the DNA had not shown up, it would not have been a technical error.

This is the cleaned up DNA from PCR 169. Dr. Kirchman cut out the DNA from PCR 169 from the gel and cleaned it up one last time. As you can see, it worked and the DNA is much... prettier :). The first tube is a control that is supposed to work every time, and it did. The test tubes with the very bright DNA are the tubes with the better-quality DNA.  The tube on the end separated from the others is a sample of DNA in a different gel so that we can make sure that there is not a problem with the gel that would cause a problem with the DNA.

Back from Break

1/16/12
I am finally back from break, so we spent the day in the lab catching up and refreshing. Dr. Kirchman had a set of books on birds and I was able to learn a bit more about rails. Rails are literally found ALL over the world, but we are focusing on the rails on the Solomon Islands. Even on these small islands, the range of diversity in these birds is amazing. The birds we are studying are categorized in the species Roviana. On the evolutionary tree of life (more scientifically known as a phylogenetic tree) Gallirallus rovianae is a decedent of the buff-banded rails or Gallirallus philippensis. Here it gets a little more tricky. A scientist (whose name I cannot recall) traveled to the Solomon Islands. A native had caught a Roviana rail, but had eaten it. However, he skinned the bird and salted it and then presented it to the scientist. The scientist recognized this to be a new species of rails, but also realized that not all of the rail's feathers had not grown in and it was therefore not an adult. Baby rail's molt patterns change from when they are young to when they grow up, the same way a duckling is yellow and fuzzy, but has different coloring as an adult.
The Roviana rail had similar coloring to the buff-banded rail and was therefore placed as a decedent of the buff-banded rail in the rail phylogenetic tree. However, since the Roviana rail is not mature, the similar coloring to the buff-breasted rail cannot be relied upon. The Roviana rail also has many similarities to a rail called Nesoclopeus woodfordi. By processing the DNA we have now collected from our Roviana rail, we will be able to determine where the G. rovianae belongs on the phylogenetic tree (I hope I'm not saying anything wrong here :O).
Our next step in our rail project is sending the DNA off to be processed and sent back to us with a nice long list of Cs, Ts, Gs, and As. However, it is cheaper for us to send a case of 96 DNA samples than it would be to have to send the 24 individual samples we currently have, but that just means I will get to spend more time in the lab! We currently have mitochondrial DNA from our Roviana rail. Mitochondrial DNA, though, is the DNA you received from your mother, and therefore only contains half of your genetic makeup (since it does not include the father's DNA). We are going to extract nucleic DNA that contains the rail's entire genetic makeup and fill up the rest of the case. I also have some exciting pictures of the results of our PCR under the ultraviolet light which I will explain above :D

Another day in the Lab :D

12/12/11
Today I was not only given another chance to 'perfect' ('form' may be a better word) my pipet skills, but also to do some aliquoting. An aliquot is basically a fraction of a whole, which in the research lab means taking a test tube of DNA and then sucking up a small amounts and plopping it in another test tube to give to another scientist for their research. I am embarrassed to admit that this seemingly simple act took up the full time of my internship. The task seemed simple: transfer 30 microliters of 30 different samples of spruce grouse DNA into 30 little plastic test tubes. Obviously, I still need to work on pipeting efficiently, but more importantly, I need a little practice writing in size 3 font legibly. My handwriting may be small, but small AND neat is a different story :). However, it was nice to get some more pipet practice in, and, from a big picture point of view, the whole process of extracting DNA is basically just moving liquids which I will hopefully be a master at by the end of the year. While before I determined being a pipet 'master' to simply understanding how to use a pipet, that definition has evolved to not only being able to use a pipet, but do so perhaps a little faster than a snail's pace. Besides my aliquoting experience, there is also some exciting news. Dr. Kirchman was able to run the filtered DNA through the gel I made last week, and while there were not the nice blocks of DNA that are found in good samples of DNA, there were stripy bands of DNA running through the gel. Dr. Kirchman cut these stripy bands out of the gel and ran the DNA contained in those bands again and to our delight, there were nice blocks of DNA in a couple of the test tubes. This means that we can continue the rail project in full force once the Winter Break is over.

PCR in a very small nutshell

oops. Day 3 and Day 4

11/28/11
Well I just noticed that I didn't make a post for my trip to the NYSM over break.  On Monday over break, I headed up to the NYSM and Dr. Kirchman and I quickly got to work. We went to the ancient DNA lab where we got some Taq gold. Taq gold is a high quality solution used in PCR. Now, what is PCR? Well, since the pieces of rail tissue we used were so small, there was not a lot of DNA in them. What PCR does is it splits the chain of DNA in half using heat(I think I am going to try to draw a picture on paint and post it).  DNA is made up of G's, C's, T's, and A's. The PCR adds ends to the DNA, and then since we know that G goes with C and T goes with A, during the PCR, G's C's T's and A's are taken from a 'soup' mixture of monomers and matched accordingly with each half strand of the DNA, forming two separate, yet identical strands of DNA.  The DNA is then cooled again so that the newly added strands can attach to each previous strand. This process the repeats as the DNA is heated again, and the amount of DNA doubles each time. We completed the long PCR with our rail DNA and then made a gel so that we would be able to see the DNA.  The gel is basically what it sounds like.  After mixing a few chemicals whose names escape me and adding a mutagen (I believe it is ethidium bromide) which allows the DNA to be seen under an ultraviolet light. After making the gel and allowing it to harder, we put the results of the PCR in the wells in the gel along with some dye.  We then ran electricity through the gel. Since DNA has a slight negative charge, it moves from the negative side of the gel toward the positive.  The gel works as a sieve, however, allowing smaller sized particles to move through the gel more easily than the larger.  However, when we viewed our rail DNA through the camera on top of an enclosed box that shone ultraviolet on our gel, we didn't see any DNA. It appeared that our rail DNA extraction had been a failure.

12/5/11
The next week, Dr. Kirchman had some exciting news.  He had tried the PCR on the thrush DNA we had extracted our first week and had found that after running the PCR on the thrush DNA, the DNA was not visible under the ultra-violet light.  He had also noticed some 'snowflakey' stuff in the tubes containing the DNA.  This means that the PCR had done something to the extracted DNA or reacted strangely to something in our quick start DNA kit we used to extract the DNA from both the thrushes and the rails. Dr. Kirchman ran the rail DNA through a filter, but the 'snowflakey' stuff remained in the tube. However, the 'snowflakey' stuff may not even be the cause of our problems and some other chemical causing the problems may have been successfully filtered, leaving DNA that would be visible under our ultraviolet light.  We spent today doing PCR with the filtered rail DNA along with other bird DNA simply to test how the PCR affect other bird DNA samples. I even got the chance to make a gel (almost) all by myself :).  While hopefully the filter solved our DNA difficulties, I will not know until next week to see if the new PCR results were successful!

my improving pipet skills :)

Solutions used in the process of extracting DNA are added in very small amounts. Pipets are used to carefully take very small amounts of liquid and put it into very small test tubes. They also have disposable caps so that you can use the same pipet with a different plastic attachment each time so that the DNA in one test tube will not contaminate the DNA in another.