Manas Kaushik Week 2

My second week at the lab was more like the routine. I came in sometime between 10-10:30 each day except Monday (Had to do early morning injections) and left between 5-6 in the evening. Besides the lunch break, I was kept quite busy every day. 

On Monday was an exception because we had to perform our injections. The fish tend to produce the most embryos earlier in the morning so we showed up at 9 at the fish facility. We wait 15 minutes each time for the fish to produce the eggs and then collect them. I was injecting my embryos with smad5. In order to inject, you have to load a glass needle with your solution using a pipette. Once the solution is inserted into the glass needle, the needle can be attached to the injecting machine. The machine allows you to lineup your needle next to the microscope so that you can see that you are actually injecting the embryo and not missing it. In order to release the solution into the embryo, there is a pedal that can be pressed with your foot. We spent almost 3 hours injecting our respective embryo before heading out for lunch. After we came back from lunch, we began sorting our embryos out. We had to identify first if they were fertilized or unfertilized. Once we identified that we had to sort out the fertilized embryos into wild-type, C1, C2, C3, C4, and C5 based on their structure. This was an important step because it allowed us to figure out how early stage patterning was being impacted by smad5. 

Embryos getting prepared for injection

Prepping for Injections

On Tuesday I tried a new genotyping master mix because last week the RAPD digest was not successful. Ben had to get some fish shipped out and today was the deadline so I had to make the genotyping work. I tried KASP genotyping instead and created the master mix. We used the same fin clips from last week because they were already dried out and DNA still remained. I took the master mix and distributed 5mL to each well of DNA. I placed the wells in the PCR machine and awaited my results. In a couple hours, the PCR was done and I could view my results. This time the genotyping had worked successfully and we were able to figure out which fish were wild-type and which were C-24 mutants. In the afternoon I went to the fish facility and separated the C-24 mutants from the wild-type. The C-24 mutants had to be shipped to another lab in Boston where they will be studied in detail. I helped Ben box the fish and send them off for delivery.

Hannah, my graduate student, was not that busy this week so I spent a lot of time helping others in the lab with some of the projects they were working on. Wednesday I helped another graduate student, Sam, fin clip around 100 fish. She need the clips for genotyping and did not want to do the fin clipping because she had fin clipped 500 fish the previous day. I offered to clip the fins for her and bring them back to the lab. This process took up the whole morning since I needed 100 fins. In the afternoon, I created the KASP master mix for Sam and distributed it into all 100 of the wells.

Genotyping results from Tuesday

On Thursday things started slowing down because people were getting ready for the July 4th weekend. I spent most of my time on Thursday in the fish facility reorganizing the fish so they were back in the system for the weekend. Certain fish are taken out of the system when they are being studied but can only last a few days out of the system before they need to be put back. Labeling was extremely important because putting fish back in the wrong tank or wrong system could screw up experiments for a long time. I made sure I always checked the labels and made sure I was putting the fish in the correct tank and system so that I would not be affecting anyones results. In the afternoon, I learned to how to use a complicated machine which allows me to pull needles. I pulled needles for injections we would be doing next week after the July 4th long weekend.

Lunch at White Dog Cafe

Not much was done on Friday since it was July 4th weekend and many members of the lab had already headed out. On top of this, Mr. Sham and Dr. Peretz were visiting. I came in at 10am as usual and began my day by helping to identify/sort Ben’s embryos from Thursday. By the time I had completed this task, it was time to head out to White dog cafe to meet up with others at Penn, Dr. Peretz, and Mr. Sham. It was nice to see our teachers and find out what my other fellow students were learning in lab. By the time I got back from lunch it was around 2:15 and Mr. Sham was coming to visit at 3. I helped Sam load a gel and then it was 3. I took Mr. Sham to the fish facility first and showed him around the area. The fish facility is a separate building from our lab which contains all the fish that we study and is where injections and fin clipping takes place. After showing him around the fish facility, I brought him into the lab and introduced him to Hannah and Ben since I was working closely with him. Unfortunately my PI was not in her office when Mr. Sham visited so he did not get to visit her. Overall I think Mr. Sham had a great visit to my lab and it was nice catching up with my fellow classmates.