Final Thoughts

April 27, 2012

Before my internship this year at the New York State Museum, I had no idea how vast the insect species is, what a PCR machine is, or how to run gels. I have absolutely no regrets in my experiences in this internship. I couldn’t have asked for a better mentor. Dr. Cryan taught me so much more than just how to categorize insect species, replicate DNA, and create master mixes. He taught me how to work in a laboratory environment, he taught me how to remain positive when experiments don’t go as planned, and he taught me to have fun while focusing on a task. In the beginning, I made mistakes creating different mixtures of ingredients into the master mix and when inserting the pipette into the grooves I made for gel electrophoresis, but in the end, I was able to complete the process more smoothly than I could ever have hoped. Dr. Cryan walked me through every step and was always patient and encouraged questions. By the time my internship ended, my accomplishments at the museum were vast and I wouldn’t give them up for anything. I consider my accomplishments from tasks as small as putting the correct DNA into the right test tube to as big as seeing my final gel after the fifth time of trial and error experiments. On my first day at the museum, I was nervous, palms sweating, knees shaking, but I soon realized there was nothing to be afraid of. It was a great experience and I wouldn’t change a thing about it. My only advice to future interns is to have fun with it and don’t be afraid to make mistakes.

Pictures at the Lab

 --> That's me preparing a master mix for the PCR machine

 --> That's me using a pipette to insert the DNA into the gels to begin gel electrophoresis

March 21, 2012 Last Day

March 21, 2012

Unfortunately, today will be my last day working with Dr. Cryan. He has been an absolutely amazing mentor, I could never have expected what a great time I have had learning and working with him. He has taught me everything from basic DNA structure to sequencing genes, to PCR machines, to gel electrophoresis. Today, we primarily worked on the presentation that all the interns will be giving at the end of the year assembly. We scrolled through some old power point slides and discussed ways to describe to people, who haven't spent months in lab learning, what exactly I've been doing. I found it especially helpful to look back on all of these long blog entries to review the vast amount that I've accomplished with my mentor. This has overall been a very rewarding experience and cannot wait to participate in another internship in my senior year!

Cancelled :(

Today, February 29, 2012, I will not be able to go to my internship because of the snow.

Can't Go

Unfortunately, on Wednesday, February 22, 2012, I will be unable to go to my science internship to do a change in the Emma schedule.

Just Another Day in the Lab

Yesterday, February 16, 2012, I went to the NYSM again. Like I said in my last post, my mentor decided to have me start all over by making the mix for the PCR but we increased the amount of Magnesium Chloride and primers. Instead of making 2 mixes for the PCR, I made 5. We worked with DNA sections 18S and 28S. I made the gels as I always do and then put them into the PCR machine. I was working with Dr. Cryan again this week. After we put the mixes into the PCR machine, Dr. Cryan took me into his office so we could look at a computer data base for DNA. He explained the process of what happens to the DNA after we do the gel electrophoresis. Since none of my gels have come out perfectly, we haven't gotten to the stage of sending off our data, but hopefully this gel will turn out right and then I can send off my mixes for real. Here's the process:
After doing gel electrophoresis, you should see a lot of glowing bands which signifies that the DNA is good, the components of the mix were done right, etc.
Then we ship off the mixes to a center that puts the mixes through another PCR to amplify and make more copies. The only difference is that in that PCR, they include some primers that are fluorescently tagged. In our PCR, we include regular A,G,C, and Ts. This other PCR has some of the regular ones but also has special A,G,C,Ts.
That place then ships the amplified mixes to another place that has a machine that is sort of like gel electrophoresis but more advanced. The gel isn't made of agar, but something different, and instead of being 6 inches long, it can be 5-6 meters long. The mixes are put on the plate and electrical currents are run through it. The special A, G, C, and Ts do two things. One, they have a stopper on the end, so when the PCR was amplifying, the DNA could only replicate until it attached a special primer that stopped it. So you would end up with
A'
AG'
AGT'
AGTA'
AGTAC'
and so forth.
The length of the chain made will determine how far down the plate the chain can travel. At the end of the plate, a laser is shot through the special primer. This causes it to fluoresce. A computer then takes a snap shot and records the color that the nucleotide glows. There is a universal code for each color corresponding to a nucleotide. I may be incorrect but I believe the the colors are
A -- green
G -- red
C -- blue
T -- black
After all of the nucleotides have been recorded by color, the computer matches the color to the letter. This data is then published and we can look at the data and move it around.
I hope the gels work next time so I can start to work on this process!

Feb 8, 2012 More Gels!!

Today as I was walking to Dr. Cryan's office, Dr. Cryan was coming towards me and in passing said that he was just called into a meeting, so he couldn't work with me today. He brought me into the lab and introduced me to Sam and Julie. Sam was talking about computer software when I came in. They explained some things about how whenever a genome for a species is completed, it gets entered into this world wide data base that anyone can view. Then, Sam went off to do his work, and Julie worked with me for the day. We ended up making two gels. The first time, I did the same things that I've been doing, making gels, inserting the DNA with a pipette, and then running gel electrophoresis. Unfortunately, nothing showed up. There was however some thing shown in the picture. Julie said it was probably excess primers that were added in the PCR. Just in case, she said we should rerun the gels and see if the same thing happens. After making a second gel, we saw that nothing appeared. This means that yet again, something was wrong in the mix. After the second gel was done, Dr. Cryan came back from his meeting. He said that next time, since we wouldn't have time today, we should use someone else's kit, someone else's station and try it again. I guess a different person tried to run it and got good results. Then we went to his instructions and compared them to mine. He had added more magnesium chloride and doubled the amount of primers. Both Julie and Dr. Cryan said that they were important changes and could very likely be the reason that my gels haven't been working properly. Next week I will start all over from using the PCR with the other person's instructions, kit, and lab station.
It was nice working with Julie today. I was able to compare different techniques and processes that the two scientists do to accomplish the same tasks. For example, Julie uses a "cross linker" to clean some of the trays before we use them. It looks like a microwave. You just place the tray into the machine, and it uses UV light to clean potential excess DNA off the tray. Dr. Cryan just uses water to clean the tray out.
All in all, I am crossing my fingers that next week's test works because we are running out of ideas. I am trying not to be discouraged, but I guess we'll have to wait and see!

February 1, 2012

Unfortunately, there will be no internship today, my mentor's son is sick :(

Hope I'm Doing this Right

Community Service Day

Unfortunately, I did not go to the NYSM today. It was the Community Service/Dr. Martin Luther King Jr. Celebration Day at Emma. I am excited to be going back next week though. As of now, no problems to report. Everything has been running smoothly.


January 18, 2012

A Brief Summary

A little while ago, Dr. Cryan gave me a report that he and Julie Urban wrote, titled "Entomologically famous, evolutionarily unexplored: The first phylogeny of the lanternfly Fulgoridae (Insecta: Hemiptera: Fulgoroidea)." The report shows the results of the first study ever of the phylogeny of the Fulgoridae lanternfly. The study was based on DNA nucleotide sequence data from five genetic loci. The report begins by explaining the uniqueness of the fulgorid species included the brilliant colors, the production of cuticular waxes, and the enlongated head function. The primary goals of the study were to reconstruct the phylogeny of the major lineages in the Fulgoridae species, test the existing classification of the Fulgoridae, and examine the evolution of the enlongated fulgorid head. Some of the tests that they do include taxon sampling, DNA extractions and PCR amplification, and sequence alignment and phylogenic analysis. In the end, the study was able to test the classification of Fulgoridae, examine the evolution of the Fulgorid head morphology, and explore the distribution patters of the fulgoridae. More research will be needed to gain a greater understanding of the fulgoridae, but this study was a great way to test old hypotheses and get a jump start on research into the fulgoridae species. When I first read it, it was all very scientific and confusing, but as I've been working with Dr. Cryan, more of this report is making sense.
Posted January 11, 2012

Something Went Wrong

Today was very much like last week. We started out by making the gel for the new DNA that we replicated in the PCR machine last week. The process was the same. We added dyes to make the DNA visible, we ran electricity through the gel and watched as the pink, blue, and purple streaks appeared. We took another picture of the gel, but the results still showed up negative. Dr. Cryan said that since the experiment had been completed successfully a while back, and since this was our second failure, there was probably something wrong with the ingredients. This means that either one of the components we added went bad or the actual DNA was messed up. Since the DNA is fairly new, we concluded that this issue must be one of the other components. In order to test the DNA and the other components, we switched out all of my ingredients for new ones, and also added another sample DNA. The new sample that we added was recently tested and was successful. This means that if the new DNA turns out positive in the photo and the others do not, then we know the DNA is bad. If all of the DNA samples turn out positive, we know it was one of the ingredients. After making a completely new mixture from scratch, we put the samples in the PCR machine and let it run. Cross your fingers all of the samples turn out positive!
Again, we finished early, so Dr. Cryan took out more insects for me to examine and categorize by species. After we finished categorizing, we placed each species in different test tubes. Each tube got filled with alcohol, I believe, and then labeled. Unfortunately, time was up so I couldn't examine more, but I'm excited to see our results of the PCR next time!

Posted JANUARY 11, 2012

Making Gels

Last time I was at the NYSM, I used the PCR machine to replicate DNA. Today, I took the samples that we replicated and made a gel with them. We assembled various substances to make the gel. One of the ingredients was this fluorescent dye that makes the DNA within the gel visible under the black light. Within the gel form, we put in combs, which are plastic comb shaped inserts that go into the gel and make about 25 grooves in the gel. We waited for 5-10 minutes for the gel to solidify. While we waited, we added this dark dye to each of the 9 samples so that they would not only be visible but also to add density. The next step took a lot of patience, precision, and a steady hand. I had to take 5 micro liters of each sample, using a pipette, and insert them individually into each groove within the gel. The gel was submerged with one liquid below it and one liquid above it. My depth perception has never been great, so placing the pipette in the exact location, making sure not to penetrate the gel was a bit tricky. After all was in place, we had to run the gel through an electrical current. DNA is negative, so by running a current that ran negative to positive, the DNA would move along with it. Because of the dyes, we could see 3 separate color lines forming. After it was done, we took the gel over to a machine that exposed the gel to the correct lighting and then could take a picture and process it through a computer. We need to do this because the picture will show bright lines on certain areas where it was positive. Unfortunately, the photo was all negative. This means that something must have gone wrong somewhere along the lines. Disappointing, but not the end of the world. In the next ten minutes, I made up new test tubes to go through the PCR machine. Next time I go, we will give the gel another shot.
Since we had an extra half an hour, Dr. Cryan went into the freezer and pulled out some insect samples. We looked through microscopes and separated the mix of all different shapes, sizes, and colors into what appeared to be categories based on species type. It was amazing to see the tiny insects under a microscope. A little weird at first, but then I got to see the different features each species had and Dr. Cryan told me what the features did. It was amazing to see some insects that had only recently evolved to have a different feature and then comparing it to the insect it used to look like.

Posted January 4, 2012

18S and 18S nDNA Replication

Dr. Cryan started off the day by giving me a little background on DNA replication and the secondary structure of protein coding versus nonprotein coding DNA. Afterwards, he showed me logs for replicating QUI54LC, I believe it was called. My first job was to replicate 18S DNA from the point a07 to bi. He showed me a list of chemicals and quantities, which we multiplied by 9 because we had 8 DNAs to replicate and one blank control. After using many different pipettes and quantities, I was able to create one test tube with all the "ingredients." Then I put 25 microlitres into each individual test tube with individual DNA. He taught me how to use the PCR machine. You place the test tubes into the machine, choose a folder, a reaction, and then let it begin. The PCR machine raises the temperature to about 95˚C then lowers it then evens out at about 54˚C. It repeats the cycle 44 times after the first cycle. We finished that one with some time to spare, so he had me do the same test with 18S on a different segment of the DNA. All in all, it was a successful day and I learned a lot about the importance of exact measurements and not contaminating different test tubes.

Posted 15th December 2011