Cross visits

Geuill showed me her in-class lab.

She showed me how to germinate seeds quickly and how to add the concentrations.

To germinate she said to:

1. Rehydrate the seeds in water overnight
2. Wet coffee filter 
3. Place five seeds in the filter 
4. Place in plastic bag and fill it with air to simulate greenhouse effect

Spring Plan

My Plan is definitely to add more, more, and more data!

My experiment will change because I will be: 

• Testing each marker
• Finding new recombinants
• Making PCR's for previous markers

My experiment will have additional data such as:

• New markers
• New recombinant fish
• New intervals
• New genes to locate for clues

Additionally, I will learn:

• How to PCR the recombinants from markers z24206 and z17291
• How to correlate all of the existing results
• How to check the genome to find how many genes are in between (http://www.ensembl.org/Danio_rerio/Info/Index)
• Process for sequencing

To make more effort I will:

• Do more research on my own
• Take note of things I learned from the Cancer Association Meeting that we attended

Reflection on PJAS

I felt strongly about my work. As soon as I begin presenting, I feel like I am not just presenting a powerpoint about zebra fish, but that I am presenting something a part of me, which is research. As soon as I start, there is no stopping me because I get passionate about sharing my discoveries. I was able to communicate my work in both a scientific and relatable way.  The judges had many questions about whether the genome of a zebrafish was published, how long this project takes, and what my next steps were, since I have no found the exact location yet.  Overall, I believe that they enjoyed the project. My next steps are to add data! I need to narrow the interval even more, since there are to many genes in between to simply sequence!

Research Paper- Proof of first page

Research Plan

2014-2015 Pennsylvania Junior Academy of Science

By Maria Isaev

Advanced Research

Ms. Cohen

Research Plan

A. Question

• What is the molecular identity of the 08bdth/spotty gene located on chromosome 2?
• How  could Spotty regulate cytoplasmic segregation during zebrafish early development?

Research Update!

This week I have cleaned out every male from the facility (since the male fish are not necessary)! I have also continued genotyping and committing my results.
Success: The cleanup took hours, but it was successful
Challenge: Euthanizing the fish is always a bad feeling:/
Question: Why is euthanizing the disposal method for fish?

Research Update!

This week I have continued to work with my new markers: z11023 and z11415.
Success: Genotyping the fish
Challenge: Remembering to use different stock solutions/ buffers
Question: If the gene is closer to z24206 and z11023, then what is next?

Abstract for JSHS

Fishing for the Gene- Narrowing the interval to identify the molecular nature of a new recessive maternal-effect gene

Maria Isaev

Central High School, Philadelphia, Pennsylvania

Ms. Galeet Cohen, teacher/ Ricardo Fuentes, mentor/ Dr. Mullins, sponsor

The 08bdth/spotty maternal-effect mutant was isolated and mapped after a four-generation crossing strategy of chemically-mutated zebrafish. By the use of Simple Sequence Length Polymorphic (SSLP) markers, the 08bdth/spotty gene was mapped on chromosome 2 between markers z24206 at 45.5 cM (centiMorgans) and z17291 at 52.4 cM. In this project, I focus on narrowing the genetic and physical interval containing the 08bdth/spotty gene by finding new genetic markers positioned closer to the mutation. The two questions I am asking are: What is the molecular identity of the 08bdth/spotty gene located on chromosome 2 and how  could spotty regulate cytoplasmic segregation during Zebrafish early development. Narrowing the DNA interval will allow me to identify the molecular nature of a new recessive maternal-effect gene, the 08bdth/spotty gene, which regulates cytoplasmic segregation during zebrafish early development. After finding recombinants between markers z24206 and z17291, I have concluded that the gene is located closer to market z24206.

Research Update!

This week I have been analyzing the results I have so far. I have concluded that the gene is closer to marker Z24206 because it had 8/240 recombinant, in comparison to z17291, which had 9.
Success: The result does not seem significant, but it is!
Challenge: Creating a chart to prove so
Question: What is my next step?

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 separate day than the actual gel electrophoresis? 

The Cloud

I have felt as though I was in a cloud once. I also felt as though I was a "pilot flying through the mist, and I lost all sense of direction". 

I felt lost when I first entered my laboratory and had to read Post doc papers on my he development of an embryo and mutations. It was all too complicated to me. I needed to understand why certain areas development at a certain time post fertilization because that explains which areas are developed, which in turn explain what mutations one zebra fish or one family of zebra fish may have. At times I've felt so flustered with the new information on genetics and cells and procedures, that I completely forget the purpose of the experiment and get too carried away trying to understand the different proteins and their functions. 

Being the youngest in the laboratory, I felt as though I was unable to bring anything completely new to the table. Just as the Ted talk explained, science prepares us to "come on stage and fail". I look back at my failure as when I first began the new experiment, because before I was able to bring results, I had to constantly repeat procedures because I made minor mistakes such as pipette errors or solution mixture contents mix ups. These were mistakes that the lab knew how to fix without starting all over, but that I couldn't.  So then as I began receiving results and recording them in my chart for each marker, I realized that I AM bringing something to the table. 

I truly believe we are part of the schema of research. I do experiments that do not have the results I prefer, and I get lost in the cloud. The boundary of the known and the unknown change my basic assumptions and have me stuck in the cloud, as the Ted Talk describes. 

I see the cloud as an opportunity because I learn how to get out of that "block". I get to brainstorm more and find loopholes and creativity. This is definitely an extremely negative feeling at first, but it becomes positive with great discoveries. 

Research Update!

This week I have created another Gel Electrophoresis. Then, I took pictures using the UV imaging device. After wards, I recorded that bands and created charts. 

Success: The pictures and bands were clear and I was able to record wild type, heterozygous, and mutant. 

Challenge: Multi-Channel pipetting the buffer into the gel is complicated because my hand must remain steady at all times in order for the buffer to reach each pocket.

Question: What other gels are made other than agarose? 

Research Update!

This week is another short week. I have been organizing my lab notebook! I have been making notes in red pen and noting pages of important results or images. I have also been pasting in new pictures taken from the previous week.

Success: Organizing and labeling important data. 

Challenge: Some scientific errors were not notes in red. 

Question: How does a scientist keep up with every observation when there is a new one for each procedure each time?!

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?