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?

Research is Key

Research is one of the main tasks when conducting an experiment. To ask more questions, and get more answers, we need to know the basics. I personally like getting my research from papers written by scientists that have already studied something similarly. For instance, this is s research paper given to me in the laboratory. This paper is about 30 pages and because the topic isn't exact, I went through to highlight what I need to know. I also read through the gene maps to have examples. Other sources I use are the Internet, books, and laboratory peers. 

Lab notebook!

   This is an example of a page from my lab notebook. This notebook contains EVERYTHING. It has every thought, question, procedure, trial, etc. 

   This page in my notebook is an example of how I graph my final results in each trial. I label the graph accordingly and then look for mistakes. Typically, I write all my errors In red. That way I am able to go back and work on them, then after wards I simply ✔️ it off. 

The Process

   The scientific process is something we have all heard of before. Beginning all the way in elementary school, we learn about the basic steps in science class-

MAKE OBSERVATIONS
ASK A QUESTION
START BACKGROUND RESEARCH
CONSTRUCT A HYPOTHESIS
TEST YOUR HYPOTHESIS BY CONDUCTING AN EXPERIMENT
ANALYZE YOUR DATA
DRAW A CONCLUSION

In reality, this process is SO MUCH MORE.
   When making observations, hundreds of questions may pop into my head. I mainly ask "Why?" and " How? " Curiosity is key to finding the answer. I personally despise not getting answers to any questions in general. With science, I find answers all on my own. Researching is the difficult part. We are all open to so many sources that we don't know where to start! But, it's important to take advantage of all of our connections. That means teachers, books, and one of our best friends- Google.
   After research all we have to do is give an opinion.
 "I hypothesize that...."






   Next is the fun part- The experiment. This could be anything, but it involves a series of trials to make sure that there aren't any mistakes on the way. (AKA Scientific errors). I've had experiences with errors in PCR. There were times when I would insert my cell plate incorrectly, label the wrong columns, or even leave units empty. Learning and redoing is all part of the process as well.
   Finally, we get the results. We analyze them and think about why they might have occurred that way. We draw conclusions and state whether or not our hypothesis was correct or incorrect. The part I love is that WE DO NOT ALWAYS HAVE TO BE RIGHT! At the end, I usually contemplate how I can relate this to things happening on a daily basis: Something huge. I also have future considerations such as how I could improve this experiment/ what other factors would I test.

   We all follow a process, but the way we partake in this process differs between every observer.

Why?

     We ask about 100 questions on a daily basis. It's in our nature to be curious and unravel our thoughts to the world. We all wonder "Why" sometimes, but how amazing does it feel to actually know the answer? I for example cannot stand living life not knowing why certain things work the way they do. There's no answer to everything, but sometimes if you put your all into finding what you are dying to know, things go the way you predict. That is why research always impresses me.  It leaves a huge impact on me because I am the one finding all the answers. I love the fact that I hold that inevitable power. It can always be unpredictable, which is the best part. Finding something that I  would have never expected gives me the need to know what happened. So why?