This week was an exciting week as I transitioned from working mostly independently to entirely independently. It involved a few mistakes along the way, but I have learned a great deal from those mistakes. Also, the tour on Friday 6/21/2024 was really exciting and fascinating as it opened my eyes to how the water we use gets filtered, which I’ll make sure to detail in the section for this day!

Monday – Iodixanol Gradient Viral Pellet Purification

Monday was a very productive day as I was on my feet the entire time barring a short lunch break! I find myself really engaged in the work I am doing here and I feel strongly towards the cause the project was inspired by. I started the day by sonicating the pellet and subsequently clarifying the lysate. To do this, I made lysis buffer by mixing (carefully as some of these chemicals are toxic) 30 mL of sonication buffer with 10.5 microliters of BME and 300 microliters of PMSF. One of the challenges for me during my time at the lab has been something that is probably quite simple and straightforward for most people: converting from microliters to mL and vice versa as well as finding the right pipette to use for each job. However, with practice, this has improved! After dissolving the chemicals needed for lysis buffer together, I prepared the sonicator. This was done by spraying the probe of the sonicator with DI water, 70% ethanol, and then DI water again, wiping in between each step wtih kimwipes, special wipes designed to leave no residue. I then put the sonicator probe into the sonication buffer that was sitting on the cell pellet Clark pelleted over the weekend. Since it was frozen rather than fresh, the sonication process was not as long. The settings of the sonicator were 7 minutes, 70% amplitude, pulse on 01, and pulse off 03. While the sonicator was running, I prepared our lab’s floor model centrifuge by grabbing the right rotor out of the fridge, putting it into the floor centrifuge, closing the lid, and turning it on; this was done to pre-cool the centrifuge to ensure that it is ready to go when the lysing is done. When the cell pellet was completely broken up and there were no traces of floating clusters of cells, I poured the lysate into a centrifuge tube that was compatible with the floor model centrifuge and balanced it with DI water in another tube, carefully ensuring they were precisely balanced to prevent any risk of injury due to unbalanced centrifuge runs. I then spun the tubes at 30,000 x g for 45 minutes at 4 degrees. Immediately after the spin was completed, I removed the clarified lysate and immediately decanted the supernatant into the supernatant tube, leaving it on ice. Then, I pipetted 2 mL of sonication buffer onto the pellet without BME or PMSF, resuspended it, and took samples of both the pellet and the supernatant for SDS-PAGE and Western blot.

Once this process was done, I prepared iodixanol solutions of 10%, 20%, 30%, 40%, and 50% using 60% iodixanol, sonication buffer, and the simple dilution formula–CiVi = CfVf. Then, I pipetted 1 mL of 10% iodixanol into 4 polystyrene tubes that are compatible with the ultracentrifuge. I then slowly pipetted all of the clarified lysate into each of the 4 tubes, 1 mL at a time, ensuring that the iodixanol cushion did not mix with the clarified lysate. Once the supernatant was pipetted, I added sonication buffer with BME to ensure that each tube was filled until the top–as Clark says: “There should be enough space in the tube that you can put the end of your pinky in.” He then spun these tubes in the Sw41-Ti rotor tube holders–I could not do this part as I do not have the necessary training to operate the ultracentrifuge. The tubes were spun at 37,500 RPM in the rotor for 2 hours and 4 degrees. Fun fact: Clark found out 2 hours was sufficient (compared to the 22 hours he used to run it for before) accidentally because he forgot to start the ultracentrifuge once. During the spin, I set up the iodixanol gradient by slowly pipetting 50%, 40%, 30%, 20%, and 10% iodixanol on top of each other in a polystyrene ultracentrifuge tube, ensuring that they do not mix and form clear lines in between each concentration. Then, I set up another tube with the same materials, but since this one would not contain any of the sample, it did not matter whether they mixed or not–it was simply a balance tube. After the centrifuge run, I removed the supernatant from one tube, and since the pellets were hard enough to not move, I poured it into a tube. I then pipetted 1 mL of sonication buffer with BME onto the pellet. I then repeated this steps with the other tubes, resuspending each of them. Finally, I pipetted the resuspended pellets on top of the iodixanol gradient (on top of the 10% iodixanol). It was very important the the iodixanol and resuspended pellet fractions did not mix. I added DI water to the balance tube and Clark spun the gradient at 37,500 RPM for 22 hours at 4 degrees in the same ultracentrifuge rotor used earlier (Sw41-Ti). This marked the end of Monday!

Tuesday – Meetings & SDS-PAGE (attempt)

We had meetings until about 11 so I got back to the lab at around 11:30. When I got to the lab, I immediately set to work on running an SDS-PAGE gel. The lanes were as followed: marker, pellet, supernatant, iodixanol supernatant, viral pellet, band 1 from the gradient, and band 2 from the gradient.

I started the day by turning on the heat block near the gel running station and heating then venting the tubes (by opening and closing). Next, I spun the tubes in the small centrifuge to get out the condensation from inside the tube. I then got an SDS-PAGE gel out of the fridge, removed the well comb, and set up the gel cassette with a buffer dam (since I was only running one gel). I placed the cassette into the gel running box and poured running buffer into it. Next, I loaded the gel with 5 microliters in each of the lanes (mentioned in the previous paragraph), repeating each sample for the Western blot. Then, I ran the gel at 100V for 15 minutes so the dye fronts tightened up on top of the separating gel. Once this was done, I increased the voltage to 180 V then ran it until the dye front ran off the gel. However, I was only supposed to run this for 45 minutes, but I ran it for an hour, so it ran too far off the gel. This resulted in the gel being unusable. I repeated this process on Thursday.

Wednesday – Juneteenth!

Spent the day working on college essays 🙁

Thursday – SDS-PAGE and Western blot

I started the day by putting the samples on the heat block and heated it for 2 minutes. Then, I vented the tubes by rapidly opening and closing and let it heat for 8 minutes for a total of 10 minutes on the heat block. Next, I removed the samples from the heat block and centrifuged them to remove the condensation from inside the tube. Then, I got the gel out of the fridge, removed the well comb, and set up the well cassette using a buffer dam since I was only running one gel. Following this, I placed the gel cassette into the gel running box, then poured running buffer into the cassette until it was about to overflow. Next, I loaded the gel with 5 microliters of marker, pellet, supernatant, iodixanol supernatant, viral pellet, iodixanol gradient band 1, and iodixanol gradient band 2. Then, I ran the gel at 100V for 15 minutes until the dye fronts had tightened on top of the separating gel. When this happened, I increased the voltage to 180V and ran the gel until the dye ran off the front of the gel.

Next, I stained the SDS-PAGE gel with Coomassie blue. I started by grabbing an empty pipette tip box and separating the front plate and back plate using a wedge gel-cracker. I placed the gel cracker into one of the bottom corners and gently pushed the back end of the gel-cracker down to open the gel. I then cut the stacking gel off and threw it away. To remove the gel from the glass, I gently slid the wedge under one of the corners of the front plate and continued pushing up slowly until the gel dislodged fully from one edge. Once this was done, I placed the gel into the empty pipette tip box. Note: Cut the gel into half and use the other half for the Western blot I will describe in the next section. I then poured the staining buffer onto the gel until it was fully covered with a few millimeters of buffer and microwaved the gel and buffer for one minute with the box unclipped. Once the gel was microwaved, I moved the box to the fume hood using heat resistant gloves, then I opened it and let it vent. Then, in the fume hood, I poured the staining buffer into the used staining solution bottle using a funnel and gently rinsed the gel off with DI water. Finally, I added destaining buffer and two kimwipes to the box and left it on the rocker until the gel is destained. I left it overnight. Finally, I put the gel into a plastic sleeve, labeled it, and scanned it.

While this was happening, I was also running a Western blot. I started this process by pouring the Western blot transfer buffer into the transfer box until it was about half full. I then grabbed some filter papers and soaked them in the box with the transfer buffer. While the filter paper was soaking, I grabbed a square plate, brought it to the gel running bench, and filled the bottom with methanol. Next, I cut a blot-sized portion of PVDF using scissors and got the blue protection paper off the blot using special forceps. I then put the blot into the methanol to activate it. To make the transfer sandwich, I placed the sponge, soaked filter paper, gel (backwards orientation), activated PVDF paper, soaked filter paper, and sponge on top of each other in that order. I then clipped it shut and placed it in the Western blot transfer box. Next, I placed a blue cooling block into the front part of the running box, put the box in the fridge, then filled it up with transfer buffer to the “blotting line” on the front of the transfer box. Next, I put the lid on the transfer box, then ran it at 90V for 1 hour. While the transfer was running, I prepared 10 mL of 10% milk in TBST. After the transfer, I removed the membrane from the sandwich and put it into a square plate with all of the 10% milk. I allowed the blot to block for 1 hour and subsequently prepared 10 mL of 5% milk with 1 microliter of antibody (6x Hist) and left it on the rocker for about a minute to mix the antibody into the solution. While the milk and primary antibody was on the rocker, I poured the 10% milk off of the blot and rinsed it with TBST until the TBST ran clear. Next, I poured the primary antibody and 5% milk solution onto the blot and incubated at room temperature for 1 hour. Once this was done, I washed the blot 3 times for 10 minutes each with TBST and made 10 mL of 5% milk in TBST with 2 microliters of secondary antibody while this was blocking. I let the antibody mixture sit on the rocker for about 1 minute to allow it to mix into the milk solution. I poured the last TBST wash off the blot then added the secondary antibody and incubated at room temperature on the rocker for 1 hour. Once this was done, I began with the next set of 3 TBST washes, once again for 10 minutes each. Next, I poured the TBST off the blot into the sink, rinsed with some Milli-Q water, then poured developing solution over the blot until the blot was covered. We usually shake it until we start seeing faint bands, but it didn’t really happen this time. This blot yielded no significant results. Finally, I clipped the developed blot with forceps and put it in the 37 degrees incubator to dry. Finally, I placed it into a plastic sleeve, labeled it, and scanned it.

Friday – Field Trip to Surface Water Plant

Today we went to the City of Sugar Land Surface Water Plant. There are four steps in surface water treatment.

1. Coagulation – Chemicals with a positive charge are added to the water. This positive charge is intended the negative charge of dirt and other dissolved particles in the water. When this happens, the particles bind with the chemicals to form slightly larger particles.
2. Flocculation – This follows coagulation. Flocculation involves the gentle mixing of water to form larger and heavier particles called flocs. Sometimes, water treatment plants will add additional chemicals during this step to help the flocs form.
3. Sedimentation – This step is intended to separate out solids from the water. The flocs settle to the bottom of the water because they are heavier and more dense than water.
4. Filtration – The clear water on top of the flocs are filtered to separate additional solids from the water. The clear water passes through different membrane filters with varying pore sizes that are made from varying materials, including sand, gravel, and charcoal. These filters remove dissolved particles and germs larger than the pores, including dust, parasites, and most bacteria. These filters, which are made out of carbon, also work to remove any bad odors. This water treatment plant used ultrafiltration, in which the water goes through a filter membrane with very small pores. These filters only let water and other small molecules through, such as salts and tiny, charged molecules.
5. Disinfection – After the water has been filtered (step 4), water treatment plants added a few disinfectants (like chlorine) to kill any remaining viruses or bacteria. However, it is also ensured that the water has low levels of chemical disinfectant so that homes and businesses are not harmed by chemical pollutants.

Below are a few pictures from the water plant:

Overall, this was a productive week and I learned a lot of new lab skills as well as valuable information about surface water filtration and why it is necessary!