Today we cast a gel in order to run gel electrophoresis. We use .6g of agarose powder, 30ml of TBE buffer and 3ul of Sybr Safe DNA stain. The Sybr Safe intercalates between the base pairs in the double-stranded DNA and fluoresces green/yellow under blue light (thus, we use a blue light transilluminator). The agarose powder and TBE buffer are combined and then heated. Once the agarose has completely dissolved into the TBE, it is poured into the gel casting tray. The casting tray has a comb in it that creates well that we place the DNA in to run electrophoresis.

All in all, the entire process looks like this.


Hopefully we can run gel electrophoresis tomorrow...

Our PCR beads finally came in! After what seems like years, we can finally follow through on our project. While Hawaii is beautiful and wonderful, its location does make it quite hard to carry out an experiment.

While waiting for the beads to come in, we prepared 13 samples by isolating the DNA we collected. With the beads in, we can now digest and amplify those samples all at once.

The PCR settings we use to amplify the DNA are:

Initial denaturing at 95°C for 5 minutes

35 cycles at 95°C for 30 seconds, 60°C for 30 seconds and 72°C for 30 seconds

Final extension at 72°C 10 min.

We isolated the ACTn3 gene by digesting it with DdeI. This enzyme cuts the strand of DNA at just the right base pair which gives us the strand we're looking for. Once we have the ACTN3 gene isolated, we can run gel electrophoresis and compare it to a DNA ladder to see if we have strands of the right length and then to see which allele each sample contains. If all goes well, the R577X fragment should be 108 base pairs and the R577 fragment should be 205 base pairs.

This is a random blog post, barely related to my independent study, but I couldn't pass up the opportunity to brag.

Our PCR beads have still not come in, I'm beginning to think I'll graduate before they do, but that is not what this blog post is about. This post is a shameless self-congratulations on winning the soccer state championship!

In case you don't care, I won't write an essay. But, in case you do, here's the article:

http://hawaiitribune-herald.com/sports/local-sports/hpa-girls-capture-state-soccer-crown


An here's a picture!

Although we ordered our PCR beads right after the second semester began (early January) by the end of January they had failed to show. They we promised to arrive within two weeks of ordering, but after a month of waiting, which was frustrating because we just wanted to start out project, they still hadn't come. We emailed the supplier, who said they had been on back order, but now were being sent. Hooray! Our happiness quickly turned to frustration as our PCR beads still failed to show. Finally, Dr. Bill called the supplier who explained that, despite being a North Carolinian company, the beads we shipped from Chicago, and that harsh winter weather (the so-called Chiberia) had slowed there arrival. Now assured that the beads, were finally, finally being shipped we settled down to wait once more.

In the mean time, a picture of our group (Me, Allex, and Justin) was featured in West Hawaii Today article about the Energy Lab being recognized as an Apple Distinguished Program (again). Here's the article:

http://westhawaiitoday.com/news/local-features/energy-lab-recognized-apple-distinguished-program

Here's the picture:

Now that we've worked out the kinks in the procedure of isolating, amplifying and running gel electrophoresis, we figured it was time to go bigger. Instead of working on a project that had been done at HPA many times, we decided to design an experiment that would test something new. We have decided to study ACTN3 gene prevalence in different sports at my school. There are two different types of skeletal muscles, slow-twitch and fast-twitch. Differences in their myosin fibers means that each type of muscle uses energy differently. Slow-twitch muscles fire slowly but use energy efficiently, meaning they can work for a long time. In contrast, fast-twitch muscles, as the name implies, fire rapidly. However, they use energy quite inefficiently and thus tire quickly. The ACTN3 gene controls fast-twitch muscle development. There are two different types of ACTN3 copies. One contains DNA that is a fully expressed in the body. The other copy contains a premature stop codon, R577X, which means that transcription of the DNA strand is cut short and a defective, but not harmful, copy of the gene is expressed. Genotype, the combination of different copies of genes you inherit, is unique in each person. Studies have indicated that different genotypes of the ACTN3 gene affect athletic performance at an elite level (Olympic and other international competitions). They have shown that high performance sprint athletes have a lower prevalence of homozygosity for the stop codon (R577XX genotype), a higher prevalence of homozygosity for the normal allele (RR) and a lower frequency of the heterozygous alleles (RX) as compared to control groups. Also, high performance endurance athletes have a higher prevalence of the XX genotype as compared to control groups. My project tests if that correlation holds true at the high school level. We will test three different groups of students. One group consists of sprint athletes from many sports: swimming, track and field, soccer, volleyball, basketball etc. One group consists of endurance athletes from cross-country running, long-course swimming, distance track and field etc. We will compare the results to a control group consisting of students who do minimal athletic activity. Beyond seeing if a correlation exists between the ACTN3 gene and high school sports we want to see if genotype affects the sports people choose to play. Does having a genetic predisposition to speed or endurance affect sport choice in high school or does it only affect success at an elite level?

Although we've identified the project we want to do, our eternal problem of not having enough supplies has once again struck. We need new PCR beads. These beads combine all the materials we need for PCR (except the template DNA and buffer) into one sphere. This includes the dNTPs used to make new strands, the Taq Plolymerase used to synthesize those new strands, along with various other elements such as magnesium needed to provide an optimal environment for DNA synthesis. We've ordered the PCR beads, so now all there is left to do is wait.

Using Polymerase Chain Reaction and Gel Electrophoresis to Test for Bitter-Tasting Ability
I am doing this project with Allex Blacksmith and Justin Pham. Allex and I began working on this project in early September and Justin joined us at the end of September. We had a couple of goals. Our first one was to inventory and organize the biotechnology supplies and chemicals at the Energy Lab. While we knew we had most of the materials we needed to run our experiment, when we arrived they we stacked in three boxes while the chemicals were in the fridge. We inventoried and consolidated all the supplies and set up a space in the Monitoring Lab. My favorite discovery was that we had three automatic micropipettes. These pipettes are much nicer than the ones we normally use in class. We also took stock of which chemicals we had and ordered the ones we needed. Our second goal was to re-familiarize ourselves with the process of PCR and Gel Electrophoresis. Our thinking was that before we began any big project we should know exactly what we are doing from experience. This turned out to be a good thing to do as we had a few missteps alone the way. Our main problem was figuring out how to create a 2% Agarose Solution that we could run DNA through. We eventually discovered how to do it, although the process involved many more steps than we had originally expected! Either way, I enjoyed learning the process and practicing creating the correct concentration of the chemicals we needed. It's cool to be able to create an end result from only a collection of chemicals in a fridge. Now that we've figured out how everything runs, we want to design an experiment that tests something much more complex. Currently we are exploring ways to sequence DNA. This mean that would would figure out the specific base pairs (A, T, G and C) in the DNA of an organism. We would have to sequence only part of the DNA of an organism because the complete genome is too huge to sequence without the use of huge computers. We are especially interested in sequencing part of the DNA of an plant that is endemic to Hawaii and then comparing it to a closely related plant that has its genome listed in the National Center for Biotechnology Information Database ( http://www.ncbi.nlm.nih.gov/nuccore). If we were certified and knew that our work was correct, we could even add the DNA sequence to the database. We could also compare the sequenced DNA to other genomes stored in the database in order to see how closely related an endemic species is to other related species.

Today is a momentous day! The rest of our materials that we need for our project arrived. We had ordered Ethidium Bromide which we will use to stain our DNA bands in order to better see them. We had also ordered a Hae III enzyme and molecular weight markers. The Hae III enzyme is a restriction enzyme. Restriction enzymes cut DNA strands at a certain point in their nucleotide sequences. In Hae III's case it cuts the DNA strand at a GGCC sequence, in between the second G nucleotide and first C nucleotide. A Molecular Weight Marker is a DNA Ladder which is the standard we run for gel electrophoresis. The DNA Ladder has strands of DNA of different lengths which we compare our DNA to when we run gel electrophoresis. Like this:

Now that we have everything we need, the experiment can begin!

This is a place where I can keep everyone updated on my independent project using Polymerase Chain Reaction and Gel Electrophoresis. These processes are used to sequence and study DNA. First you get a sample of DNA. Saliva is a common way of acquiring it. Then you create a lot of copies of the section of DNA you want to study by using a Polymerase Chain Reaction. Basically you put your DNA strands, a lot of DNA bases and an enzyme that can put the bases in the right order into a vial. You then heat the vial up and let the enzyme do its job. The enzyme basically creates thousands of copies of a single strand of DNA. Then you use Gel Electrophoresis to sequence the DNA. In Gel Electrophoresis the DNA strands are sorted according to molecular weight. This means that the longer the strand, the farther up the tray it will be. We do this because a longer strand means that there are more nucleotide bases. The more nucleotide bases there are, the more proteins that can be sequenced from that strand and the more stuff a cell can do if it has that certain strand of DNA.

Right now I'm waiting for all the various enzymes and chemicals to arrive at the Energy Lab. Once those are in, the project can begin!