12 Jul Eleanor Rosen, Briney lab, TSRI- Week 3

Hello everyone, week three, I’m officially over halfway done, and am so sad. I love getting up each morning and seeing what the lab has in store for us. This week was really fun. We did a lot, and got to feel like really a part of the lab, doing cool things. This week the lab, and us learned that we are willing to do anything, because it’s all new and fun, so we did a lot of work that other people in the lab have been putting off, or they needed to get done while working on their bigger projects.

Monday, July 6,

Monday morning we got to the lab, and watched a video on PCR cloning. We then were told our schedule for the week, and started the cloning process. This meant making a substance known as the master mix (or what belle accidentally nicknamed the megamix), which is a mix of an enzyme, DNTP, buffer, and pure H2O. This was then sectioned into trays with the cells for the hantavirus.  Because antibodies have both a heavy and light chain, we had to do both, and create primers both forwards and backwards. It made it really confusing, but we managed to portion all the mix, DNA, and buffer into the tray. After that we put it in the thermocycler for 45 minutes. We took them out, put them in another tray, added another buffer and put them back in the thermocycler. After that we began work on our main project for the week, this is a project for Nathan, where we will be doing Midipreps for 39 different antibodies, twice, for Lassa virus, and Ebola. Each antibody has a heavy chain and a light chain, so we have to do the process for both chains. Monday we started by prepping for the heavy chain reactions. This meant taking 39 beakers, filling them with 125ml of LB media, and then adding in antibiotics which will kill everything but the cells we want, then adding in our cells to begin growing. Once we had all these flasks we went on a little adventure around the lab to find enough room on the incubator shaker plates to fit our media. Once we got them all loaded up we went home for the day, we left the lab proud of our work for the day, and excited for the rest of the week. We walked over to UCSD to head home with Stella and Kaila. After getting home we went to IN-N-OUT in celebration of our AP scores coming out (we all passed!). After dinner Zoe, Jennie, and I went grocery shopping for the week, and we then headed home and relaxed.  

   Our flasks in the shaker plate (only 1 of 3 used)

Tuesday, July 7,

When we got to the lab on Tuesday we did a zoom with Benjamin who explained the history of Hantavirus, and the different infection types and symptoms. It was really interesting and now we know more about the virus we are working on. After the zoom we set up for the first 39 Midipreps we would do. The midipreps basically mean taking about 125ml of liquid and getting it to about 500µl of DNA, and buffer.  Tuesday was heavy chain day, we got the prep kit unpacked, and began the work. We began by spinning down each media solution to get a cell pellet. With the pellets liliana and I began adding all of the P1 mixture in and vortexing them. While liliana was vortexing, I began the process of adding P2, and P3, then getting all the cell debris out. Lastly we added the binging buffer in, and I gave them to Belle who was in the vacuum, which washes them, and leaves just DNA. Lastly, I added the elution buffer, and we centrifuged them. The last part of the process meant taking the finished product over to the Nanodrop to measure the concentration. About 51% of our DNA had a good enough concentration that they didn’t need sequencing, the ones that were probable we then sent in for sequencing. The ones with bad concentrations will need to be redone again. We ended the day by preparing the solutions for the next day. After work we headed home where we all relaxed for the evening.

Midiprep filtering out cell debris.

Measuring concentration on the Nanodrop.

Wednesday, July 8,

Wednesday marked the official halfway point of our internship. And it was a great day. When we got to the lab we barely had time to put our bags down when Nathan told us to put on our lab coats. We found that one of our mixtures never grew, and we had a duplicate, so we only had to do 37 samples. We set to doing the same process as Tuesday, and worked for 4 hours straight. When we tested the concentration of the samples we were disappointed to see that only 14 of them worked, though we were assured that is normal. When we told Nathan he said if they are above 100ng/μL they should still work, so we sent those in for sequencing as well. After lunch we did the Gibson assembly for the PCR we did on monday, we were all so exhausted from the morning that we couldn’t stop laughing at basically anything, which was entertaining. We then bleached the beakers from the morning, and set those out. We were sitting at our desks when Nathan came up to us and told us he had very important work for us, and it was something he had never done before. He then handed us 3 pieces of paper which had the QR code for the sequencing on it, and he told us to draw dinosaurs on the back. Turns out the company they use for sequencing optionally asks for people to draw dinosaurs on the back of the paper, and if their kids like them you win a prize. So we ended our day in the lab drawing dinosaurs. After work we went on a public tour of the Mormon temple. We figured, when in Rome, I mean when do you get the chance to visit a temple of a highly secretive religion. It was an interesting tour, we decided they only showed us what they wanted to. Later Jennie, Zoe, and I took Patillas to the dog beach, and got ice cream and poke on the way home. It was a fun outing, and a great way to end the day.  

Our dinosaurs

Thursday, July 9, 

Somehow the three of us made it out of the house all wearing the same color. No one noticed till we got to the lab and Terrance asked us if it was planned. We were then told no less than 6 times if we were aware that we had worn the same color and asked if it was on purpose. A really funny experience for the day. When we started working we began combing the heavy and light chains of the antibodies from tuesday and wednesday, this meant a lot of math, and then changing pipettes each time, then we centrifuged them. We then went back to our desks where terrace had me add to my dinosaur drawing from the day before to make it more nerdy, so we added a tRNAsaurus-rex. We then emptied out our beakers we filled with bleach the day before, to prepare them to be picked up for cleaning. We were then released for the day so we took the trolley home. I made dinner, and Zoe, Kaila and I went on a walk to get boba.

Our antibodies and math. 

Friday, July 10,

Today we got off work early so we went to the Torrey Pines State Beach, it was a really nice start to the day. When we got to the lab we were informed that it would be a slow day, so we took the opportunity to catch up on some training, and work on our blogs for the week. In the afternoon we shadowed another person in our lab Raiza Bastidas, in doing transfections. This meant counting cells on a plate, we all got to try this, you mix in a die that colors the alive cells, and then put it in a counter, which tells how many cells there are, and how many are dead. Then we got the media with the cells in it and created a new mixture as well, we then filtered some media and added the DNA in. This was finally added to the cells, and set it to grow. After that we headed home, where Belle and I read in the yard. After everyone came home we had dinner and enjoyed the evening. 

Raiza doing transfections

Saturday, July 11,

We had a slow morning where I made pancakes and bacon for everyone. Then we went to the mall for the day. We all divided up, where Zoe and I learned we are bad at shopping but pretty good at eating. After the mall we spent the night at home, we made wonton soup as a family, and ate dinner watching the Argentina vs Switzerland game.