Manas Kaushik Week 4 and 5

To start week 4 I was introduced to Matias, another graduate student in the lab. Since Hannah, my graduate student, was going to be away at a conference, I was going to be working with Matias for the next few weeks. On Monday he showed me the project he was working on and where all his materials were kept. In the afternoon, we went down to the fish facility and he showed me where all his fish were and gave me two tanks that would be mine. Today we discussed some genotyping which I was going to do for him. He told me it was some complexing genotyping and would require longer than a week but he thought I was ready for the challenge. There was not enough time to start the process today so we decided it would be better for me to start tomorrow.

One of my tanks

On Tuesday, I went down to the fish facility and began fin clipping. Each tank had around 50 fish so my plan was to fin clip one tank today and the other on Wednesday. The fish I was working with had just been born a couple of weeks ago so they had never been fin clipped. I fin clipped one tank in the morning and placed all the clipped fins in methanol. Once I got back from lunch I vacuumed out the methanol and created a rapd digest. The rapd digest would make sure the fin clips were dried out so the DNA could be taken out. I did not start the pcr process yet because I wanted to finish clipping both tanks and do them together. 

On Wednesday I continued fin clipping. I finished the second tank of fish so now I had clipped around 100 fish. Once again I vacuumed the methanol out and created a rapd digest to dry out the fin clips. I let both sets of fin clips sit in the incubator overnight to make sure the fin clips were completely dried out before I started extracting the DNA for pcr. 

Fin clipping

I came in early on Thursday so I would have enough time to do pcr and run a gel. Unfortunately the fish I was working with could not be genotyped using the kasp method so I had to use a longer process. I made a 3% gel using agarose and buffer. I had to be careful while making the gel because I was using ethidium bromide which can be toxic. The 3% gel would be thick so it would take longer for the bands to travel to the positive side. Once I made the gel I started loading it. Since I had all the wells lined up in rows of 12, I was able to use a multichannel pipette. This allowed me to pipette 12 wells at a time. There was 25 wells in each gel I made so I placed the ladder in the middle so it would be easier to analyze the bands and had 12 sets of DNA on either side. 

Fin clips stored in methanol

On Friday I took images of the gels and did not get the results I expected. Matias and I spent multiple hours analyzing what could have gone wrong because we did not believe it was a human error. We made a plan for different sets of primers we would use in which some cases two primers were needed and in others three primers were needed.

Analyzing some of my results

Week 5 was frustrating at times but it taught me that science does not always go the way you imagine. I ended up running the pcr and gel for Matias’ fish five times and our results were still not conclusive. I tried different primers as instructed by Matias but still we were not able to differentiate between the heterozygous fish and the mutant fish. Both of them showed two bands in the results and we could not make assumptions without knowing for sure. The fish had been out of the system for a while and needed to be put back so they could be fed. Unfortunately we felt the DNA was beginning to go bad after a week of testing so we decided to move on from this. A combination of some bad DNA and some bad primers prevented us from getting the results we hoped we would get. Although a full week of hard work had gone to waste, I still learned new techniques and was able to consistently run a pcr and a gel.