Purpose

The 08bdth /spotty maternal- effect mutant was isolated and mapped after a 4- generation crossing strategy. It was mapped on chromosome 2, by the use of simple sequence length polymorphic markers between markers z24206 and z17291. 

In this project, I will focus on narrowing the genetic and physical interval containing the gene by finding new genetic markers positioned closer to the mutation. These maps guide scientists to mnay genes that are believed to interact to bring about more common disorders of diseases. 

So...... This is essential because....

• 70% of protein- coding human genes are related to genes found in the zebrafish and 84% of genes that are known to be associated with human disease have a zebrafish counterpart. 
• Zebrafish and humans both share the characteristic of being vertebrates.
• Some zebrafish mutants are known to develop and duplicate certain conditions and diseases common to humans.

Research Update!

This weeks goal was to select fish from a brand new designated tank, tank F144. I continued genotyping more fish. 

Success: Muti- fish fin clipping! I've improved on time and was able to genotype many more fish. 

Challenge: This tank contained more than 48 fish, therefore only half was genotyped. 

Question: Why does it take longer to anesthetize the smaller fish? 

Procedure Description

I have recently learn how to multi- fish fin clip. (Original name I have created).  This is a step- up from the previous fin clipping because it enables me to clip 3x quicker.

To multi- fish fin clip:

Preparation-

Prepare a well plate filled with 100 uL of methanol.

1) In the fish facility, create a solution of 0.02% Tricaine (10 mL of 0.4% Tricaine solution, 190 mL of system water. 200 mL total)

2) Remove fish from designated tank, into a larger mouse box.

2) Set up single boxes and label according to each well.

3) Add the Tricaine solution with water, into a separate single box.

4) Select 3-5 fish using a net, and leave them in the Tricaine solution to anesthetize.

5) Remove one fish at a time, and transfer to a petri dish. Using a toothpick, separate the tail and clip a part of the fish's fin with a razor blade.

6) Use the toothpick to put the tail in the designated well.

7) Put the fish into the corresponding single box and repeat with the other fish.

The purpose is to fin clip more fish in a day, by saving time. I feel confident doing it, since I have had months of practice fin clipping one fish at a time. This fits into the next step for the day, which is the Lysis step. 

Research Update!

This week is a short week, therefore I will be focusing on organizing the laboratory freezing and creating my individual box of materials for my PCR reaction. This will make it easier and quicker to begin my PCR. The individual box will include a lysis buffer, stock solutions for each marker, F+R solutions for each marker, 10x PCR Buffer, DNTP solution, etc.

Success: A scientist must always stay prepared to ensure that they have no experimental flaws, which is why organization is key to an effective project.
Challenge: There are over 100 different solutions in the biology lab freezer! Finding every necessary sample size solution took focus.
Question: Why must the taq solution, necessary for PCR, be kept and lower temperatures than the rest?

My Equipment!

I have many, many materials being used in the laboratory. Each set of procedures includes various materials, which are all vital to my experiment. 

When identifying recombinants and narrowing the physical interval that
contains 08bdth/spotty gene, I begin with fin- clipping, therefore I must use

• 12-48 Zebrafish from a designated 08bdth tank

• 1 large mouse cage for the 48 fish 

• 1 tank net, 1 blue net to retrieve and select the fish 

• Individual smaller mouse boxes labeled depending on the amount of fish and wells

• Tricaine Solution and individual mating box to anesthetize the fish 

• Tea strainer to retrieve fish from Tricaine

• Petri Dish to clip the tail in 

• Razor Blade to clip the tail 

• Tooth pics to separate the tail 

• 96- Well Plate to put each tail clipping in

Then I begin Lysis for DNA extraction, for this I need: 

• 96-well plate with methanol and fin clips

• Pipette tip connected to a vacuum source to remove methanol 

• 55 ºC incubator to incubate over night 

• Lysis Buffer to lyse the cells 

The next day, after incubation, I must Create Master Mix. For the master mix, there is a working solution for designated markers. For this I need, 

• Stock Solution and working solutions for each marker, for the PCR

• DNA for amplification

• Centrifuge to spin down the well plate with the solution for PCR

• PCR machine used to amplify the DNA

After the PCR reaction, I must create a gel. For the gel electrophoresis, it is essential to have a: 

• Gel chamber for seperation and analysis of DNA, RNA, and protein along with other fragments, based on their size and charge. 

Lastly, I must use the BIO RAD Ultraviolet imaging camera to take pictures of the gel, revealing base pairs. 

Research Update!

This week I have been taking more pictures of my gel, recording band results and repeating with the other marker. I also labeled the pictures and pasted them in my lab notebook. 

Success: I did not to reprint any pictures because they were all tuned to a fine resolution. 

Challenge: The camera on our floor was out of service, so I used a different one which was more complicated to function because the settings and buttons were different. 

Questions: Would pictures be the same resolution if the pictures of the gels were taken on a seperate day than the actual gel electrophoresis? 

Research Update!!

This week at the laboratory, I have been analyzing some of my images from my genotypes. To analyze these images, I must count the number of base pairs, and from there note whether they are mutant, heterozygous, or wild type . 

Success: Identifying whether the bands indicated mutant, heterozygous, or wild type. 

Challenge: Working the translucent lamp in the Bio Rad UV camera was difficult and required multiple practice prints of pictures for the correct resolution. 

Question: What does it indicate if the band does not appear?