12 Jul Liliana Almanza Mendez – Scripps Research @ Briney Lab, Week 3

Insane it’s the 3rd week of my internship!! So lets just go right to it:)

Monday, July 6th

Today we did Polymerase Chain Reaction (PCR) cloning with Hantavirus still continuing on with Benjamin’s project. To recall our purification with the first sample of Hantavirus was not successful, so Jonathan ordered a different primer to test…

To begin the process Jonathan resuspended the samples, then we made the Master Mix; which included buffer, water, and an enzyme.With the Master Mix made we then proceeded to add it into all 42 wells. When this step was complete we added each of the corresponding primers which Jonathan had already labeled on sheets, I’d say this part was the most challenging because some primers worked for multiple antibodies. To make it easier and minimize errors Belle ended up tracking and checking off each well while me and Ellie pipetted, which turned out to be a great method because we only messed up one time. Once everything was assembled we put it into the ThermoCycler (machine heats and cools at different times to activate all the different enzymes in the reaction) with the suggested times and temperatures in relation to the primers Jonathan chose, which in this case was running for 47 minutes. Once this was done we were supposed to by protocol check the ”concentration” of the samples to see if it was worthy of continuing to the next step in the procedure, but Jonathan had faith and just told us continue. So we added ExoSAP – FT, this is to eliminate everything but the DNA, and back it goes into the ThermoCycler.

Nathan then introduced us to a projected he had started last December, but failed to succeed with, this was mostly due to it being such a large project. In his project he is using 39 monkey antibodies and trying to figure out their effect with an antibiotic in correlation to its ability to neutralize.  Today we only did the heavy chain tomorrow we will begin the light chain. The process began by filling up 39 250mL flasks with 125mL of LB media, then we labeled all of our flasks, then we added 125µL of antibiotic to each of the flasks. We then became a NASCAR pit crew!! Belle and I were opening and closing the caps of the flask so Nathan could scrape the frozen antibody from the tube and eject the pipette tip inside each corresponding flask, while Ellie was handing Nathan the antibodies. We decided to do this because if you let the frozen antibodies thaw out they could lose their ability to preform said reaction. To end the day we had In-N-Out for dinner, which was delectable per usal.

Tuesday, July 7th

Let me start off by saying that today was a very long but productive day in the lab… I will not be explaning in detail the DNA prep as I did so last week and it’s the same prep as a mini but on a larger scale. For a quick recall we do this to have DNA samples ready for further research and studies a.k.a their the starting block. It’s very important to do this protocol correctly because if you don’t you won’t have any DNA to purify or transfect with!

Nathan set us up with all the necessary materials to begin all 39 Midipreps  for the heavy chain which he guided us through the protocol once, while giving us some tips and pointers to make the process more efficient. Slowly but surely we got rolling and made a system, I poured the DNA from the flask into the centrifuge tubes, Nathan centrifuged, Ellie and I vortexed and prepped while Belle vacuumed. Once I finished transferring all of our bacteria growth I made 5.5L of LB media. It was so much easier this time because Nathan gave me the master mix powder and all I had to add was water. Last week if you recall we made 17L of LB media and we actually followed a ”recipe” and had to mix multiple powders instead of just using the master mix, all is well though because that gave us measuring practice:) After we finished prepping all of the DNA we went over to the NanoDrop to check the concentrations levels. As usual we did a system so I was adding 20µl of each sample, while Belle labeled the concentration on the tubes and Ellie recorded on a sticky note. As expected the samples we forgot to add binding buffer too did not have a great concentration, but the rest had great concentrations some even reaching 7,000µg. Since we had to stay longer at the lab, we got picked up at 5 instead of 4 which felt so odd. All is good though because I went to adoration and called it a wrap for today.

Wednesday, July 8th

Today is going to be very short because we did what we did yesterday, but minus 2 so 37 Midiprep’s for the light chain. Again Nathan set us up with all the necessary materials, although he had a very busy day ahead of him so he told us we would be alone but if we were to have any questions to ask Jonathan. We had the same system as yesterday expect Belle centrifuged and I vacuumed. Once we prepped everything we went over to the NanoDrop to check concentrations. These numbers did not look at all, most were very low:( We then added a 10% solution of bleach to the media flasks and let them sit for 30 minutes so they could be cleaned later. It was already going to be 3:30 when Nathan came over and said ” I have something very important for you guys to do and I’ve never done it before” and man did we get scared. Turns out he was going to send out our DNA for sequencing and on the paper it says to optionally draw a dinosaur and if their kids like it you can earn a prize.

Thursday, July 9th

When we sat at our desks this morning, Terrance came over and asked us if we had planned to match and wear all maroon. This was funny because we hadn’t even noticed we wear all matching and neither did anybody at the house. Since then we had six other people in the lab asked us if we planned to match, which was so funny.

Nathan then told us that even if our concentration from yesterday weren’t very high most of them where good plasmids. Nathan also gave us the DNA light and heavy chains to match up, so I did that while Ellie did the calculations with the formula Nathan provided and Belle labeled. Once we finished half of them I began mixing, I mixed in the amount Ellie got using the formula, so every measurement was different. Multiple pipette switches later and tips, we finished and began centrifuging them so the samples cold be sterile and ready to go under the hood for transfection.

Friday, July 10th

We had a great morning today! It all began by making coffee at the house and two breakfast burritos. We were getting dropped off earlier because Jennie had to be at the airport by 9 so we decided to get dropped off at the glider port. We were planning to sit around like we usually do, but on second thought we decided to hike down to the beach. Honestly best choice I have made since we’ve been here, it was so peaceful but fun in the right amount. On our hike back up we decided to take the goat path which was pretty difficult but also worthy of experience.

Once we got to lab I began the ATD Training and BSL-2 Safety Training, these trainings are required for all personal that are working in BSL-2 labs. We then shadowed Riza whom was doing a transfection for Terrance. She didn’t really know what she was working with herself, but they were proteins and in a very big amount (6.5-7million cells).

<– This is Biological Safety Cabinet (BSC)

Saturday, July 11th

Me and Kalia walked down to the trolley station about an hour earlier than the others, because she didn’t want to be late to the movie theater. This gave me a little extra time to shop, which is always appreciated. Once everyone else arrived we regrouped and began conquering every store in our path! I even went to Melo Melo a store that sells coconut flavored jelly jars. I went with their banana Nutella which was really good, but the texture definitely threw me off a bit. To end the day we did a family dumpling night for dinner and watched the World Cup game.

Thanks for reading! I hope you enjoyed it, because I know I did…

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Manzanita 