Lab update!

This week I have been learning and practicing to gel. I used my results and calculated the amount of solution and amount of agarose I need. I add a very toxic chemical known as Ethidium Bromide and heat up the solution. The solution then turns into a gel. I pour it into the gel tray and add 12 mL of the loading buffer. I will be working on reading the gels and taking pictures of the bands soon! 

Success: Creating the solution 

Challenge: Pipetting the loading buffer 

Question: What does the loading buffer do? 

Lab update!

     Lately, I have been working in the fish facility of the laboratory.

I have been genotyping! I gather fish from a specific mutant tank named: 08bdth. I anesthesize the fish, clip their tails, and run the lysis buffer. Then I create master mixes for each stock solution: 

Afterwards, I add 1 mL of DNA and 19 mL of each master mix. PCR Is the final step then I begin to run the gel! 

Success: Fin clipping! 

Challenge: Creating master mixes

Question: What does the lysis buffer do ?

Who am I working with?

     I work with a Post- Doc named Ricardo in the Cell and Developmental Biology Laboratory in the University of Pennsylvania. He is from Chile and has worked with Zebrafish there for about 10 years. He gained interest in cytoplasmic movement in Zebrafish embryos. He came to the University of Pennsylvania in the year 2000, studying biology and cellular movement. He also learned to appreciate genetics and expand his knowledge on the cytoplasm. That is most likely what makes research the most enthusiastic- applying previous knowledge to learning new things. Since he has already had previous experience with Zebrafish and cells, he had advancements for the Cell and developmental biology laboratory. The passion I observe throughout this entire laboratory inspires me to want to be a scientist. Most substantially, this inspires me to be a scientist in this exact field. Outside of the lab, they live their regular lives with their spouses and children. The undergrads focus on schoolwork, while the post docs and other grads join various clubs or talks around campus, and bike about everywhere! Ricardo has been in this specific lab with Dr. Mullins for two years and he has two left! Then it's back to Chile or even Germany! 

Paper Report!

 Source:

Dosch, Roland, Daniel S. Wagner, Keith A. Mintzer, Greg Runke, Anthony P. Weimelt, and Mary C. Mullins. Maternal Control of Vertebrate Development before the mid blastula Transition: Mutants from the Zebrafish I. Diss. U of Pennsylvania, 2004. Philadelphia: Department of Cell and Developmental Biology, 2004. Print.

     This paper explains why and how early genetic mutations occur. The mutant I am working with suffers from a very early embryonic mutation. This also means that the mutant dies 8 hours post fertilization.

Questions?

50 Questions pertaining my experiment: 

• 
  

1. What mutant am I working with?
2. How many phenotypes are visible?
3. How many Wildtype Zebrafish are in each tank?
4. Why do only the females matter in the beginning?
5. Why am I studying this specific mutant?
6. What purpose does Methanol serve in each well?
7. How long can the Zebrafish survive in Tricaine?
8. How long does it take for a fin to grow back after clipping?
9. Does the fish die as soon as part of the tissue is accidentally clipped?
10. How much DNA is used from the fin snip?

• 
  

1. What are the genes of each Zebrafish?
2. What are the genes of each tank on an average?
3. How many are homozygous?
4. How many are heterozygous?
5. How many are recombinant? 
6. How do we know when we are closer to the marker?
7. How many genes per marker?
8. How many genes per map?
9. How many markers per map?
10. Once one gene is found on a marker, am I closer to the next gene or marker?

• 
  

1. What is PCR?
2. What is denaturation? 
3. What is annealing?
4. What is extension?
5. Why is the standard temperature 94 degrees Celsius? 
6. What does the lysis buffer do?
7. How is lysis tested?
8. How much of lysis is needed to run a test?
9. What is Taq? 
10. What is the 10x PCR buffer's purpose?

• 
  

1. What does the dNTP solution do?
2. Why is the gel in 3%? 
3. What is agarose?
4. What is the purpose of using agarose for the gel? 
5. What is the purpose of Ethidium Bromide?
6. Why is Ethidium Bromide highly toxic?
7. What is the loading buffer?
8. How do the molecules become negative?
9. What is 100bp DNA ladder?
10. How does the gel make the poles attract?

• 
  

1. What is each band on the DNA ladder?
2. What is the measurement between each band?
3. What does one band indicate?
4. What do two bands indicate?
5. How is a wildtype visible?
6. How is a recombinant known?
7. How long does the DNA sit for?
8. What is the maximum time the DNA is "good" for?
9. How do I know when I found the closest distance between each marker?
10. How many genes per marker?