Today, I prepared DNA samples for
my mentor. When last week, I prepared the buffers to be inserted in the
capillary machine, this week, I held the responsibility of producing the main
ingredient: the DNA samples.
Using different types of 76mers –single-strand
DNAs that contain 76 bases–, I first created a mixture and put it in a sample
tube. This tube contained a combination of 5 microliters of each 76mer. Next, I
allotted to 10 separate tubes, 2 microliters of this very mixture. Because it would be the same product, if this
mixture was simply put in the capillary electrophoresis machine, one would get
a graph with about the same peak levels for each DNA. Given the fact that our end goal is
to find out the specific location of the particular 76mer within the mixture,
my next step was to add 0.4 microliters of each unique 76mer into the 10
separate containers. This 0.4 microliters is called the “spiking sample” as it
will increase the concentration of that particular 76mer in that particular
tube so that when inserted into the machine and read, the graph will indicate
the precise location with a peak that is higher than the other 9 polymers
present in the mixture. After all this, I added Formamide—a denaturing
compound—used to separate potential dimers/hair-pins created between the
single-stranded 76mers. This step is necessary as the DNA sample has to be kept
single-stranded in order for the bases (adenine, guanine, thymine, cytosine) to
base pair with the DNA. These base pairs will then help differentiate the speed
at which the DNA sample hits the cathode end of the capillary electrophoresis
machine. Later on, my mentor will use these very samples to figure out the
sequences of each of the 76mers based on the graph that will be produced from
the capillary electrophoresis machine.
All in all, I am looking forward to seeing how the samples I prepared for the first time actually turn out next week!
This is a nice blog post, Christi. You create a complete picture of your daily activities, which is easily understood. I am very curious about how this relates to your bigger project.
ReplyDeleteHi Christi,
ReplyDeleteWe had a really fun and enlightening conversation last week. I learned a lot from what you said and understood that the way you are sequencing DNA is faster than that of the gel one we did in biology class. It was also interesting to learn that you differentiate the bases by looking at the peaks the computer generates instead of the bands on the gel.
:)
Looking forward to what you'll do next~
Yvonne
Thanks, Yvonne.
DeleteChristi,
ReplyDeleteYour internship sounds pretty complicated to me so kudos to you for doing a great job. I was a little confused by the final goal of your internship before our meeting, but you explained to me that you are trying to make gels that are able to separate DNA fragments of different lengths more accurately. It was really interesting to hear about you using gel electrophoresis in your internship because I was learning about DNA extraction techniques in AP Bio at the same time which made it all the more fun to be able to talk with you about your internship. Keep up the great work and it sounds like a lot of fun!
Thanks, Kelsie.
DeleteChristi, your blog to me is especially interesting because it really seems to correspond to a lot of stuff we have done in AP bio! I was familiar with gel electrophoresis from our lab experiments in biology, but you definitely helped me understand more about capillary electrophoresis through your detailed blog posts and your explanation in our meeting. In this post, your use of electrophoresis to locate a specific 76mer is really interesting in that it reminds me of how in my internship we use a mass spectrometer to locate certain dimers or trimers in a protein solution. It's cool how all of our internships seem to connect in little ways like that!
ReplyDelete