Manas Kaushik Week 3

Week 3 did not have much going on to start off the week because we received a four day weekend for July 4th. I spent Saturday-Tuesday in Florida with some friends from school before returning to Philly on Wednesday. In Florida I went to the beach, hung out at the pool, and ate lots of good food. 

Spent July 4th weekend in Clearwater, Florida

When I returned to the lab on Wednesday, there was a new undergrad who joined the lab. I was put in charge of getting him all setup. I introduced myself to him and he told me his name was Jack. I found an empty desk for him and he put all his stuff down. Next, I showed him around the lab and introduced him to all the members. The rest of the morning I spent teaching him basic techniques for the lab like pipetting and identifying/sorting embryos. Ben, a graduate student, took Jack in the afternoon and introduced him to the more complex techniques and helped him continue practice the basic techniques I taught him. Although I had only been in lab for two weeks I realized how much I had already learned. In the afternoon, I created the solutions I needed for injections on Thursday. After I created all the solutions I pulled some needles to make sure I had enough. The needles were very easy to break so I wanted to have plenty of extras. I brought all the materials down to the fish facility so everything would be ready for injections. Injections must be done earlier in the morning so I did not want to be running around looking for materials early in the morning.

Got moved to a new desk

I came in at 8:30am on Thursday to make sure I had enough time to do all my injections. I wanted to get through at-least 1000 embryos so I had enough samples. I went to my favorite microscope (the one I try to use every time) and put all my materials down. I went to the fish room and paired multiple sets of males and females together so they would lay eggs. I waited 15 minutes and then collected the eggs. It is important not to set too many fish at a time because all injections have to be done at the one cell stage and they start developing quickly. I loaded my needles with bmp7 and smad5 and began injecting. I injected 250 embryos with bmp7. The bmp7 should keep all the embryos wild-type. I injected 250 different embryos with smad5. Smad5 should change patterning so that there are no wild-types. The embryos should take the form of either C1, C2, C3, C4, or C5. The other 500 embryos were first injected with smad5 and then injected with bmp7 to see if they could be “rescued”. Luckily I only broke two of the needles so I had plenty to continue injecting tomorrow. All these injections will help us figure out the role bmp has in early stage patterning/formation. I waited about 4 hours after injection to start identifying/sorting the embryos. At the 4 hour stage I would be able to see which embryos are fertilized and unfertilized. It is important to get rid of the unfertilized embryos because they can mess up the data as they were not affected by the injections. Once I got rid of the unfertilized embryos I put them back in the incubator so they could continue developing. Since we were going to inject on Friday as well, I created more smad5 and bmp7 solution. I took them to the fish facility and reorganized the fish. Fish typically lay well only once a week so I had to use different fish from the same tank. 

Egg Cycle for Zebrafish

I came in early once again to inject. I got my favorite microscope again and was ready to repeat the process. Injecting can be intense because it requires a lot of focus and can take a couple hours. It is very easy to lose track of time while injecting. I injected close to 500 embryos today to add to my other set of embryos. In the afternoon, I finished identifying the embryos I injected on Thursday into Wild-type, C1, C2, C3, C4, and C5. Since the embryos I injected today were not ready to be completely sorted, I just removed the unfertilized embryos that could potentially mess up the data. I planned on finishing the identifying of the embryos next week.