Week 3

Hello again! I hope you guys have been enjoying your internships and your Rodeo Break.

On Monday, Feb. 17th, I started to run gels with my mentor Naomi. We ran a small gel with a primer called up2 which was a primer that is a little before the target segment of DNA that we're looking at. Later, we scored some older gels that the AGI team made and took pictures of and decided to re-run some of them. So we ran large gels with PCR plates that were made before. I got to insert the DNA using a 12 channel multi-channel pipette. At first, it got a little tricky trying to make sure I got all the PCR products inside each pipette and that I put all of the product inside each well and the pipette kind of gets unbalanced as you pipette out the PCR products. So I also did some practice pipetting with water and a plate.

12 Channel Multi-Channel pipette

After seeing the results of the gels we ran, we decided that the primers that were used before may not be working such as the down1 primer (which is further down the DNA from the target segment). The bands seen in the gel represent whether the DNA have the O. glaberrima or O. barthii allele or both. Also, the ends of each gel are the base pair ladders (I believe for this gel we used the 100 bp ladder). You could also see how there were some gaps in the gel...there might not have been DNA in there.

Large gel with down1 primer (it might be kind of hard to see)

Therefore, I got to prepare new primer solutions. Mostly, it consisted of making tons of dilutions and dealing not only with molarity but also nano-moles and micro-molar solutions. I felt like it was chemistry math (mainly the dilution equation: M1V1 = M2V2) on a whole different (smaller) level. These new primers were called qSh1-1, qSh1-2, sh4-b1, sh4-b2, sh4-b3-R. So instead of just handing out the primers already made for us like in our Capstone Bio class with the mitochondrial DNA primers, I got to actually prepare the forward and reverse primer solutions. Although it was just simply diluting and pipetting, I'm glad I got to know how things were made from the beginning.

On Wednesday, I observed my mentor preparing the PCR with the new primer solutions I made. After loading the PCR mixes into the PCR machine, I went into my second lab meeting. The more I see these lab meetings, the more I think, "Good thing we got lots of practice presenting during our capstone classes." Although these lab meetings are more of discussions and informal presentations, I could notice things that went well and things that didn't. But anyway, the main topic for this lab meeting was on new data relating to SNPs and imputations. I'm not sure what those terms mean and I think the other members of the AGI team didn't quite understand either, so a lot of time was spent trying to understand how the data was computed. At the end of the meeting, there was a discussion about rice and arsenic (which thankfully I had some background info in). It seemed as though there was more arsenic in the food supply in general than what everybody thought. Although there were jokes about how this fact might negatively affect the rice project the AGI team was doing (since they said rice was the answer to the 9-billion people question and could feed the world). However, it also seemed like the arsenic was coming into the food supply not only from natural causes such as volcano eruptions but also from human activities such as using pesticides/herbicides, mining, and burning coal and oil. So there were also discussions of how this issue of rice and arsenic can also provide a new path for research since the team was already interested in starting a new study on the roots of rice plants, they could study the arsenic levels and how the arsenic gets into the rice plants/roots (so we possibly may have a new research study underway in the next few months).

After the lab meeting, another member of the AGI team, Dario (who has been helping me a lot) and I ran a medium gel with the PCR products. I got to make the medium gel from scratch with about 1 g of agarose and 70 mL of TAE buffer (which is a common buffer used in gel electrophoresis and for those who are curious what TAE stands for -- it is Tris-acetate-EDTA). The results of the gel showed that one of the rice species (O. glaberrima) was highly concentrated so it did not clearly show the results of the other two rice species (O. barthii and the man-made heterozygous mix of the two species). So, I prepared another PCR but diluted the DNA. (I also accidentally diluted the O. barthii when I wasn't supposed to -- this just shows how important proper communication is in a lab -- but it was fine). I then continued to prepare a new PCR mix and loaded it into the PCR machine.

That was the end of my week! Hope you guys have another great week! I look forward to your next posts! See ya'll then!