Day 4 - Butches among fish?
Day 4 - Butches among fish?
My mentor told me that we couldn't do Western Blot today because we could not get fish embryo to do it. He just asked me to follow whatever Shen Qian and Hong Ni were doing. So I went to their bench to join them.
The schedule for that day was to do RNA isolation and Genotyping the fishes. The purpose of Genotyping is to determined whether the fish is genotypically male. The difference between female and male fish in medaka is at their back fin. Male medakas have discontinuous jagged back fins whereas their female counterparts have continuous smooth back fin. However, not all medaka with discontinuous jagged back fins are males, some of them are genotypically females (so I called them butches, female with male appearance).
After preparing our RNA sample from medaka's ovary and ran them for RT-PCR, we went to the aquariums to get some supposedly-male medakas to be genotyped. What we did was to cut a small part of their tails, homogenize it to extract the DNA, perform PCR and run gel electrophoresis. If the fish is genotypically male, two bands will be observed from the gel run, but if the fish is actually a female fish, only one band will be observed. We took samples from 34 fishes and put them back in separated aquarium.
After that, we went back to our lab to homogenize the sample using a pestle. The pestle really looks like a large size cotton bud. It is white in colour and has two bulging ends. Crushing 34 small pieces of fish tails were quite tiring, luckily we split our jobs. After we homogenized the sample, we added celects, a reagent that will bind to everything that will interfere with PCR. Then we performed PCR to each sample.
While waiting for PCR to finish, we run a gel to visualize our RNA isolation sample. The result was very disappointing as I only can see a smear on my lane showing that my RNA has been completely degraded. Most probably I was not careful enough while handling the reagents... Sigh....
In the end, we decided to run our gel for genotyping on the next day as it was getting late.
My mentor told me that we couldn't do Western Blot today because we could not get fish embryo to do it. He just asked me to follow whatever Shen Qian and Hong Ni were doing. So I went to their bench to join them.
The schedule for that day was to do RNA isolation and Genotyping the fishes. The purpose of Genotyping is to determined whether the fish is genotypically male. The difference between female and male fish in medaka is at their back fin. Male medakas have discontinuous jagged back fins whereas their female counterparts have continuous smooth back fin. However, not all medaka with discontinuous jagged back fins are males, some of them are genotypically females (so I called them butches, female with male appearance).
After preparing our RNA sample from medaka's ovary and ran them for RT-PCR, we went to the aquariums to get some supposedly-male medakas to be genotyped. What we did was to cut a small part of their tails, homogenize it to extract the DNA, perform PCR and run gel electrophoresis. If the fish is genotypically male, two bands will be observed from the gel run, but if the fish is actually a female fish, only one band will be observed. We took samples from 34 fishes and put them back in separated aquarium.
After that, we went back to our lab to homogenize the sample using a pestle. The pestle really looks like a large size cotton bud. It is white in colour and has two bulging ends. Crushing 34 small pieces of fish tails were quite tiring, luckily we split our jobs. After we homogenized the sample, we added celects, a reagent that will bind to everything that will interfere with PCR. Then we performed PCR to each sample.
While waiting for PCR to finish, we run a gel to visualize our RNA isolation sample. The result was very disappointing as I only can see a smear on my lane showing that my RNA has been completely degraded. Most probably I was not careful enough while handling the reagents... Sigh....
In the end, we decided to run our gel for genotyping on the next day as it was getting late.
Labels: Genotyping, Medaka, RNA isolation
posted by Melinda Leonie @ 7:03 AM
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