Samuel Cohrs – 2023 IWU Freeman Asia

Research

I have officially finished all of my markers. The end of the week involved running some markers with GC buffer that did not amplify or weakly amplified and were highly showed a high concentration of Dimers. It yielded improved results for about half of the markers I ran with it. Thankfully, the markers continued to amplify after I changed the water. I must say as grueling as it was to trouble shoot I learned more in those two weeks than probably the rest of the internship combined in lab. I became much more methodical in my procedure and I think it improved my results overall as well. Not only that, I also learned many concepts about the procedures while troubleshooting and trying to figure out what was going in. I also gained a serious appreciation for my lab notebook. I started adding every time I diluted new dNTPs, the stock I got them from, and methodically recorded every aspect of my procedure that changed at all. Next week, I will be working on my poster and will run a couple more markers to try and get some better results with some of the markers I have already run.

Social

This week Dr. Kim was gracious enough to take the whole lab out for lunch to a Korean BBQ restaurant. Being an all you can east restaurant, me and AJ made sure to get our moneys worth. Also, later on in the week I ended up playing Ping-Pong against Dr. Kim. This was completely unplanned and resulted from us seeing each other in the gym. I never thought I would sweat so much from a couple of games of Ping-Pong. It was a really fun time though. I have been working out with a PhD student, Rowell, from one of the labs our lab works closely with, and it has been awesome to have a workout partner. The weekend was full of activities too. Friday we went out to a karaoke bar where the poor souls in the room had to hear me attempt to sing but, it was a good time. After we went to a local bar with some fellow lab mates. The next morning we went to Tagaytay, the location of the smallest active volcano on the planet and has the unique feature of being a volcano in a lake, and the inside of the Volcano is another lake. Sadly, it rained all day so we ended up going to a mall and shopped. First though, we got some really good Filipino food by lake Taal, the location of the volcano.

After El Nido this was a pretty slow week. We got back on Monday night. Tuesday was back to a normal day of work and… while I was gone Ate Grace figured out why everyone’s PCRs weren’t amplifying. It was the nanopure water. Honestly, I am not sure how I missed this seeing it was really the only variable left. I used different water but it was all from the same source and was taken at the same time. Looking back in my notes it was pretty clear that was the only variable that hasn’t been truly isolated. Either way, we are luckily back on track in lab. Other than that there is not anything new that has been going on research wise.

The weekend was pretty calm too. Friday and Saturday were full of relaxing and getting some work done. Sunday we went to a mall in Calamba which was roughly a 30 minute long Jeepney ride. At the mall, we decided to watch Mission Impossible in the movie theater which we all enjoyed. We also went out to dinner and got some Korean BBQ. Other than that the week was pretty routine where I work go to work, head home, go to the gym, sleep, and repeat. I wish I had more to report on But there was nothing to thrilling.

El Nido

The only way I can see doing this trip justice is by giving it its own section. El Nido hands down has been the most beautiful place I have visited. Crystal clear water surrounded by limestone cliffs full of bright green flora.

We left for the airport Thursday night at around midnight. Our flight was at 4 AM and we wanted to be early for our flight. After we arrived in Puerto Princesa, hopped in the van we rented and the driver sped off. What we thought would be 6 hours filled with sleep was anything but. We arrived 4 hours later, no sleep to mention, and all maybe just a little car sick. Despite the uncomfortable ride, we did arrive at our Hostel 2 hours earlier than expected. Getting to the hostel from the street was definitely interesting. You walk down a very steep flight of stairs, probably about 30 of them, to the beach, where you are supposed to walk across to get to the hostel( the beach was the only way to access it as there were other buildings behind it). The complication was that we came right as high tide so when we got to the bottom of the steps the only place to walk was water. Hey, it adds to the adventure. So, we stumble inside, check-in, put our things in the corner, and wait for our rooms to be ready. I immediately went to the water and went for about an hour-long swim. Once we moved everything into our rooms we went back upstairs and started socializing with the other guests. Within maybe 10 minutes we already made friends with people from all over the world. Singaporeans, British, Spanish, and of course the local Filipino staff who were amazing. Each night there was some type of contest where the winner would win a free breakfast. A group of us decided to go out in El Nido with some of our new friends which ended with us at a Billards bar with everyone having a great time. The next day was the boat tour which was easily a top 5 most surreal experience of my life. Just being on the boat and seeing the limestone cliffs towering over us was incredible. The boat tour consisted of 5 stops. The first and last were both similar where we played sand volleyball. Knowing me I finished the game with most of my body covered in sand. Another stop was snorkeling where we saw beautiful reefs and I even saw a sea anemone with a clownfish hiding inside. The other stop was lunch which didn’t consist of much other than eating on the boat. The stop I found to be the most incredible was the big lagoon. This was an area of water (fully transparent water might I add) which was surrounded by huge limestone cliffs. We kayaked in and the coolest part for me was the small cave we went into. The whole trip was full of stunning views that never got old. The rest of the trip really involved socializing with other people. I could on for days about the views we saw but the pictures are the only thing that comes close to doing El Nido justice.

Work

The lack of amplification still has not been solved. It was a week full of painstaking troubleshooting with little to no results. The only way I have been getting amplification is if I make each marker’s PCR mix separately which, while time-consuming, is better than no result. The strange thing is it does not seem to be a problem with my procedure because multiple others have used my markers and also did not get any results. The lack of results is happening to everyone in lab to some extent as well so it

All variables

DNA- We tested the DNA with the nanodrop and the concentration was good. The samples sometimes do amplify as well so the DNA is present.

Buffer- Tested multiple times with multiple different stocks and did not seem to change results

dNTPs- This was what I believed was the issue at the beginning but I have used so many different dNTPs without any correlation to when the product amplified or not

Taq- Unlikely. One of my lab mates tried my Taq and got results.

Procedure- Unlikely- No amplification regardless of the person running the test

Nanopure water- Unlikely- Never been a problem in the past and have used different stocks of the water

PCR machine- Unlikely- has worked for others

Loading Dye- I got excited because I thought this was the problem last week but sadly does not seem to have an effect.

Gel Electrophoresis- Unlikley- Has worked for everyone else

Pictures

Social

This week social wise started off with social hour with a large group of interns. Social Hour consists of one of the interns sharing a presentation about their home country. This month it was Pakistan. I was really intrigued by the vast sites filled with beautiful views. We also were able to try traditional Pakistani foods which were excellent. Later in the week, the IRRI interns played volleyball with other interns which was a blast. During the weekend we hiked Mount Makiling which was definitely an experience. The mountain was full of leeches which were unpleasant, to say the least. By the end of the hike, I made the decision that just not looking till the end was better and pretending they weren’t there. The view was incredible though and the flora alone was amazing. Toward the top, it was a lot of climbing but everyone made it through okay. After the hike we went back, I took a very long shower and took a much-needed power nap. We went out with Hunter and Sam later that night and showed them some of our favorite spots in the town.

Research

Well, it happened. The dreaded moment all my Professors have warned me about. The point where your experiment doesn’t work and you have no idea why and just have to keep trying. The whole week I got no amplification of my markers. I was having trouble the previous week so going into this week I threw everything I could think of (shout out to Ate Pat, Ate Grace, Ate Lots, and all the others for all the help) to fix it. First, I used the Nanodrop to check the DNA concentration which came back normal. Our first test yielded very confusing results or lack thereof. I tried to isolate every variable in some way. I tried to separate DNA and different markers that were used in a previous experiment and “knew” worked. I isolated each possible PCR mix variable and tested the different PCR components I used. I then used different DNA samples that were used for my markers and tested each of those with previous markers that worked and the markers that haven’t been working. I also had another experimental group within this level which also tested PCR products. Just to be sure there were no errors in protocols or how I carried out the procedure we reviewed those as well. No errors were discovered in the procedure. I ran the PCR and then the gel. The results…. no amplification. One of the others in the lab said the same thing happened to them despite the Nanodrop reading adequate concentrations and extracting new DNA resolved the issue. This would not explain the other DNA not amplifying, though. So, the following day I went to the glass house and retrieved new samples. This consists of finding the second youngest leaves on the plants of the 16 species I am comparing. It also involves sweating through the clothes you have on. I brought the samples back to the lab and then returned to my dorm to shower and change. After lunch, I extracted the DNA and ran the PCR/gel which, once again, had no amplification. This test was with all markers that I knew worked but were markers within my experiment. The next day I came into lab and ran the experimental setup I tried on the first day with the new DNA. I also used a different PCR machine. Once again, no amplification. In the afternoon, I ran it once again but this time I used different loading dye because at this point, might as well try it because nothing else worked. In this round, I set up the plate to differentiate between 3 DNA sets, and 3 PCR components, one of which was premixed by another lab member and they used that day which worked. The results. Everything amplified! I was very relieved while also a bit annoyed that the issue was the easiest thing to fix and the one variable we didn’t consider. This was what I thought to be a big lesson for me to read and record carefully because I missed the fact that I used a new loading dye the first day the issue started happening. Yet, I went into lab the next day, and… no amplification. I ran a couple of PCRs and got some amplification but not to the extent where the results could be scored. We will see what we can do next week and hopefully resolve the issue. I will say, I have learned a huge amount this week about the experimental process and understanding how to isolate variables which as grueling as the week was, I am happy about.

Pictures

Social

On Tuesday, I went to Batangas, more specifically a beach in Batangas, with some of the people from my lab. It was an awesome experience consisting of great food, some beach competitions, volleyball, a banana boat, and a really bad sunburn. It was nice talking to the whole lab in a more informal setting and just learning how many of them are at the point they are at now and where they are going. Friday though, the IRRI crew went to Manila. We stayed in a great hostel. I met two guys, one from Melbourne and the other from Manchester who have been traveling for a few months. It’s always awesome to meet people from all over and just chat, at least in my opinion. The Brit and the IRRI crew went out on Friday night to see what Manila nightlife was like and it was a very fun time. Saturday, we went to the Mall of Asia where my first stop was Shake Shack (as awesome as Filipino food is, it was nice to have some American comfort food). We walked around for a little bit and then headed back to the Hostel. It was a really fun weekend and a nice getaway from work.

Research

This week was an interesting one. I loaded my first 384 well PCR plate which left me with a very sore shoulder the next day. This may have been one of the most nerve-racking experiences because if my ample did not amplify that would be an 11-hour work day down the drain. An already tedious process was exacerbated by more wells, different pipetting techniques, different PCR mix volumes, and loading 4 gels at once. My favorite mistake was to load the DNA and forget to leave a space for the latter. The former became the latter in the procedure after I think the second time I made that mistake. The start of the week went great because I was able to validate 24 markers a day instead of 12. We had some trouble with one of the species not amplifying but we are fairly certain that’s due to not having a complimentary sequence to the marker instead of an experimental error. I am really starting to understand the inner workings of the reason for validation and how markers work as well. At first, I understood markers were primers but the exact correlation left me a bit confused. Now (after scoring the markers) I understand that the markers are within a specific gene where an InDel occurred (that part I knew) but, Only certain species possess this InDel causing that amplified product to be a different length. The InDel itself is only an identifier and not necessarily the most important mutation of a specific gene. That’s what I realized this week. The end of the week was a sad day. Everything went great, or so I thought, and then I went to check my gels for amplification. Nothing. Not one amplified. I finished the run at 3:30ish, showed my lab mates, sighed, laughed a little, and called it a day. Hopefully, it was the PCR products and my next PCR will amplify as normal, or else next week is going to be a week full of troubleshooting.

Quick Summary

Week two started with a trip to the botanical gardens of Mount Makiling. The flora and fauna were amazing and being surrounded by nature was very relaxing. One of the most interesting things studying biology has done is notice how different forests are in different climates, something I would be oblivious to before. When we went to the gardens we definitely were not prepared for the dense forest hike we ended up doing which was quite difficult, or at least I thought so. The sounds of cicadas overwhelmed the forest and were deafening at times. As we walked through the dense flora we saw butterflies fly by that were the size of small birds and insects that you can see in the picture section of the blog. Every once and a while the cicadas would pause allowing us to hear the birds calling in the trees. The workweek was full of activities. I feel like I am starting to truly get into a rhythm in lab which now that I say I will most likely have a major mess up tomorrow. We also attended the 6-month progress meeting where the labs in our group discussed what they were working on and the different labs gave feedback on the work. The weekend was full of activities that I will be sure to talk about in the social section.

Social Aspect

My lab has been very very welcoming and the whole lab treats each other like family which makes work that much more enjoyable. every couple of hours I will be sitting at my lab bench working and I’ll hear “Sam Coffee” which is one of my colleagues making sure I have my caffeine fix and plenty of snacks throughout the day. Let me emphasize the never-ending flow of snacks. Me and one of my lab mates, AJ, have become very close friends and whenever the IWU interns go out AJ is right along with us. He is from Mindanao, the southernmost main island of the Philippines. I have made many other friends along the way whether that be interns or someone else I encounter. A big group of interns and some visiting scientists went to Villa Escudero, an old coconut farm that is now home to a museum and other activities to educate visitors on traditional Filipino life. One of the activities consisted of eating a lunch filled with traditional Filipino dishes at the foot of a waterfall which was an interesting experience, to say the least. After returning back to IRRI, a group made up of interns from different master’s and undergraduate programs went out to a local bar where the owner had the same welcoming energy as everyone else we came across.

Research

This week was full of long days in the lab which I really am enjoying. I am about a third of the way done with validating my markers via PCR and gels. The first gel I ran during the week felt perfect and I thought it was going to be a perfect amplification which It would have been if I remembered to add the ladder. I also had my first marker not amplify after running it twice and next week I will add some GC buffer to the PCR mix which hopefully will prevent self-annealing of the amplified region, a relatively easy issue to fix if that is what is causing it. If that does not work I will have to play around with the PCR temperatures which will truly be a slightly more educated approach than guessing and checking (this will also require me to run only one marker at a time instead of six so I am really hoping the GC buffer will do the trick). that about sums up the research aspect of the week, it was a lot of PCRs and Gels but I definitely have been adjusting my protocols to try and make the process more efficient.

Photos

Quick Summary

A lot has happened this week as it is my first week. To begin, I had my flight to the Philippines which was a 32-hour trip with a layover in Istanbul. During my layover, I went on a boat tour of the city and saw the incredible architecture Istanbul has to offer. When I arrived in the Philippines it was night and we drove from Manila, where the airport was, to Los Baños, the location of my internship. The next day, after a very long night of sleep, we had an orientation where all the interns in the Philippines were present. We went to a restaurant following the orientation where we not only got to try out traditional Filipino dishes but also a few words in Tagalog we learned earlier that day. The following day the Students at IRRI had an orientation for the institute and I was able to meet my lab supervisor, Dr. Kim. The next day was when I started my research. While I am here, I have been given the task to validate InDel Markers, a genetic tool that can be used to aid in the identification of certain genes that could play vital roles in the sustainability of rice as a crop.

Social Aspect

Socially, everyone that I have met has been extremely welcoming. From the lab, to IRRI staff, to others I have met the environment is very welcoming. Me and some of my lab colleagues went and got Jollibee this week, the fast food staple of the Philippines. Think of KFC, Mcdonald’s, and pasta all in one. That’s Jollibee in a nutshell. The Freeman interns at IRRI have also gone out as a group a couple of times trying the local foods and venturing around town. The local dishes have been excellent. Our lab has “mandatory snack times” where I get to try snacks from everybody’s home countries (which I think is extremely cool). Stay tuned for the Balut taste test (feel free to search Balut if interested).

Research

As stated above, I will be validating InDel Markers which allows for the discrimination of wild rice species and cultivated species alleles. Specifically, I will be working with the CC genome. I will utilize PCR and gel electrophoresis to analyze and validate the markers. Wild species of rice have survived for millions of years, many times in suboptimal conditions. Many of these wild rice species not only contain genes that provide resistance to biotic and abiotic pressures, but also they contain certain genes which improve yields. On the first day we tested two markers, the main goal was to familiarize me with the procedures for DNA extraction (which is done by retrieving rice leaves in a greenhouse that may be the warmest air I have ever breathed), PCR amplification, gel preparation, and gel electrophoresis. Sadly, something was wrong with either the buffer or the dNTPs used for the PCR mix on the first day, and the whole lab except one get not get gel results. On the second day, we switched out the buffer and dNTPs and got a great result in the gel. This would not have been possible without the guidance of Ate Pat, the master’s student who allowed me to dive into the research on the first day I was in lab and taught me the ropes. While my project is not to identify specific alleles to utilize in cultivated species, having this “reservoir” of InDel markers will allow other labs to easily access and utilize genes from wild rice species which likely will result in profound improvements in rice crop sustainability and nutrient makeup.

-This paper published out of the same lab I am working in is where I learned many of the statements made in the previous paragraph and can provide a more in-depth explanation of the lab objectives as a whole.

–https://www.nature.com/articles/s41598-021-88533-9

Photos

Some Traditional Filipino dishes we tried after our orientation

The view from IRRI

IRRI group went out to eat

Hello!
My name is Sam Cohrs and I am a junior biology major. This summer I will be in Los Baños, Laguna completing an internship at the International Rice Research Institute (IRRI). I am very excited for the opportunity to use the knowledge I have gained in my labs and lectures in a way that has direct real world implications. Last summer, I completed an internship in Kisumu, Kenya which was an amazing experience and I am excited to have another opportunity to be immersed in a culture which I hope to learn much about while I am there.