Hi Everyone!
I hope your week has been going well!
I have some exciting news.
The gels have finally started to work! My mentor, Sampath Rangasamy, and I developed a new PCR Protocol where I tested out four different systems. There were samples that would be mixed with magnesium chloride and a FAST HS dye, without magnesium chloride and FAST HS dye, with magnesium chloride and Robust HS dye, and lastly, without magnesium chloride and Robust HS dye.
Basically, these four different sets allowed me to visualize which system would be the best in producing results for genotyping when I would upload the PCR samples into a gel. I found two that worked best: without magnesium chloride and Fast HS dye and with magnesium chloride and Robust HS dye.
Hopefully, once I conduct more samples I will be able to quickly genotype large sets of samples at a time.
I have also begun research alongside my project where I will be working with zebrafish. I have only just begun my training but mainly I will be injecting a dye into the zebrafish eggs to see if in 72 hours, the fish test positive or negative for the gene we are searching for. In the next week I will gain more knowledge on the exact research I will be conducting and will be able to share more details soon.
Other than that, things have been moving at a fast pace! I have started to work on my article with another intern. We are trying to have the rough draft ready by the end of the next month. The article will mainly be following the same guidelines as our poster presentation. Along with the article, I have been spending some time outside of the lab working on CITI Training. In order to work with the fish, I have to complete training on zebrafish online through a bunch of modules.
I am extremely excited for you guys to hear more about the work I'll be doing in the upcoming weeks.
Stay tuned! :)
Pooja
Friday, February 26, 2016
Friday, February 19, 2016
Blog 2: 2/19/16
Hi again!
I just wanted to update you all on this last week.
We are still trying to find the core problem with our gels. I repeated the PCR protocol multiple times this week and casted several gel electrophoresis. However, only once, did a single band show up for one of the samples.
I reviewed past notes and images the lab had to see the procedures that were conducted which led to successful gels and learned that they stopped working when we got new enzymes in the lab. I also found out that when the gels were working, the PCR was set to a different program than the one we currently use.
These are the two possibilities as to why the gels are not working right now. On Monday, the lab will be getting new enzymes and hopefully then, we will be able to genotype our samples.
But I have some exciting news as well!
Last summer, another intern and I held a poster presentation on the research we had conducted over the course of a few months. Now, we are given the chance to write an article on our results. It should be done by the end of the month!
I am excited to begin this new process and to go to the lab on Monday to see how the samples turn out!
Thank you for following me along the journey of this research process!!
Hope you all have a great weekend,
Pooja
I just wanted to update you all on this last week.
We are still trying to find the core problem with our gels. I repeated the PCR protocol multiple times this week and casted several gel electrophoresis. However, only once, did a single band show up for one of the samples.
I reviewed past notes and images the lab had to see the procedures that were conducted which led to successful gels and learned that they stopped working when we got new enzymes in the lab. I also found out that when the gels were working, the PCR was set to a different program than the one we currently use.
These are the two possibilities as to why the gels are not working right now. On Monday, the lab will be getting new enzymes and hopefully then, we will be able to genotype our samples.
But I have some exciting news as well!
Last summer, another intern and I held a poster presentation on the research we had conducted over the course of a few months. Now, we are given the chance to write an article on our results. It should be done by the end of the month!
I am excited to begin this new process and to go to the lab on Monday to see how the samples turn out!
Thank you for following me along the journey of this research process!!
Hope you all have a great weekend,
Pooja
Wednesday, February 17, 2016
Obstacles
Good Morning Everyone!
For the past few days in the lab, we have reached an obstacle in our research. As stated in my previous post, I have cast several gels and have run multiple PCRs. But recently, the gels have not been producing efficient results.
The confusion lies in why. Earlier last week, when I followed the original A140V PCR Protocol and ran the original "Main: A140V-HF Program" (the program that is running on the PCR machine), two gels worked. Now, when I am following that same protocol, I am not reaching conclusive results. My mentor and I have altered the protocol and even the program slightly each day in hopes to find the genotype of the samples.
Today, I will be following the original protocol one more time with a slightly altered PCR program. Fingers crossed for it to work!
Pooja
For the past few days in the lab, we have reached an obstacle in our research. As stated in my previous post, I have cast several gels and have run multiple PCRs. But recently, the gels have not been producing efficient results.
The confusion lies in why. Earlier last week, when I followed the original A140V PCR Protocol and ran the original "Main: A140V-HF Program" (the program that is running on the PCR machine), two gels worked. Now, when I am following that same protocol, I am not reaching conclusive results. My mentor and I have altered the protocol and even the program slightly each day in hopes to find the genotype of the samples.
Today, I will be following the original protocol one more time with a slightly altered PCR program. Fingers crossed for it to work!
Pooja
Friday, February 12, 2016
Blog 1: 2/12/16
Hello all!
Monday morning, February 8th, marked my first day back in the lab at TGen. I was welcomed by several exciting tasks and projects which I will begin to pursue in the next following months.
The day started off with me running a 1.8% agarose gel electrophoresis. Now, I know what you must be thinking, what exactly does this mean or what is the purpose of this gel? A gel electrophoresis separates DNA fragments based off of its size. An electric current then moves the DNA molecules across the gel.
The above image shows what a gel electrophoresis image looks like. The first column of bands is known as the DNA ladder. This is the set that all of the other bands will be compared to in order to genotype the DNA. Using the samples we have in the lab, I ran over 5 gels this week with only 2 being successful. Gels may not work for several reasons: the temperature may be too hot, the enzymes may be degraded, the gel may not have run for a long enough time, etc.
Using a 100 base pair DNA ladder, I was able to genotype several samples distinguishing if they were heterozygous, wild type, or mutant. If heterozygous, two bands will show up in the image. If wild type, there will only be one band on the bottom. And if mutant, there will only be one band on the top, which is exactly what we are searching for and are still yet to find.
Throughout the week, when I was not running a gel, I was running a PCR. Testing about 7-8 samples at a time, I followed the lab's new PCR protocol. After the PCR samples were done, I would upload them into a gel, completing the cycle.
In the next upcoming week, I will be furthering my research project by learning to conduct western blots, by working with mice models and even tissue samples. I will also begin to intern at the Center for Rare Childhood Diseases (C4RCD) where I will see the clinical aspect of the research in the lab. I am very excited to see where this research will take me and to eventually find out more about this severe disease.
Bye for now!
Pooja
Monday morning, February 8th, marked my first day back in the lab at TGen. I was welcomed by several exciting tasks and projects which I will begin to pursue in the next following months.
The day started off with me running a 1.8% agarose gel electrophoresis. Now, I know what you must be thinking, what exactly does this mean or what is the purpose of this gel? A gel electrophoresis separates DNA fragments based off of its size. An electric current then moves the DNA molecules across the gel.
The above image shows what a gel electrophoresis image looks like. The first column of bands is known as the DNA ladder. This is the set that all of the other bands will be compared to in order to genotype the DNA. Using the samples we have in the lab, I ran over 5 gels this week with only 2 being successful. Gels may not work for several reasons: the temperature may be too hot, the enzymes may be degraded, the gel may not have run for a long enough time, etc.
Using a 100 base pair DNA ladder, I was able to genotype several samples distinguishing if they were heterozygous, wild type, or mutant. If heterozygous, two bands will show up in the image. If wild type, there will only be one band on the bottom. And if mutant, there will only be one band on the top, which is exactly what we are searching for and are still yet to find.
Throughout the week, when I was not running a gel, I was running a PCR. Testing about 7-8 samples at a time, I followed the lab's new PCR protocol. After the PCR samples were done, I would upload them into a gel, completing the cycle.
In the next upcoming week, I will be furthering my research project by learning to conduct western blots, by working with mice models and even tissue samples. I will also begin to intern at the Center for Rare Childhood Diseases (C4RCD) where I will see the clinical aspect of the research in the lab. I am very excited to see where this research will take me and to eventually find out more about this severe disease.
Bye for now!
Pooja
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