Today at The NYSM I finally got my ID badge with my picture on it, so I
can go up to the lab on my own once I get there. My mentors, Ms. Urban and Mr.
Adams explained in great depth the concept of PCR and how it works. PCR
(Polymerase Chain Reaction) is basically the process of DNA replication; it all
happens considerably fast thus, the chain.
We watched a video of how DNA denatures and how different polymerase
attach and replicate a whole strand of DNA; Ms. Urban used the website
"DNAIC.org" to show me the videos. After the videos Ms. Urban explained how the
Nucleotides are arranged in the DNA and why it’s possible to replicate them.
She even explained how the DNA looked like, from shape, the Hydrogen bonds
holding the double strand together to the Copying enzyme (Polymerase). She
showed me pictures of the Phosphate group in the Nucleotides, and how the
Nitrogenous bases are reverse on one side of the DNA.
With that concept in mind Ms. Urban and Mr. Adams
informed me that they had ordered a Primer for me to use for my very first
experiment. She explained that the primer NAD6-F and NAD6-r (*review my blog
for more on this region of the DNA); would be mixed with pure water, PCR
Buffer, MgCl2, dNTP’s (A, C, T, G in mix) and T-aq Polymerase to
replicate the DNA. She also explained the role of most of the ingredients, the
MgCl2 is just to stabilized the electronegativity of O2 in
the DNA base. In addition she and Mr. Adams explained where and why they use
the T-aq Polymerase. Apparently the guy who discovered it, found it in a
bacteria in a hot spring. For anyone who knows anything about DNA, the hydrogen
bonds can be broken using high temperature, so everyone was fascinated to find
out how this particular bacteria survived in such high temps without denaturing. They worked on
this bacteria and extracted some of its Polymerase, the Thermosaquaticus or T-aq.
Scientists have been copying this Polymerase ever since and are trying to make
a synthetic version of it. So this T-aq Polymerase simply adds the Nucleotides
to make a new, second strand on the DNA after separation.
After all this very exciting conversation we head
in to the lab and Ms. Urban showed me how to prepare my very first mix for PCR.
We used a 12-template holder but made only 6, 5 for the experiment and the 6th
as a control. When preparing an experiment we use a sheet of paper that has the
“recipe” for the mix. We used the ordered primers, (NAD6-f and NAD6-r) and our
Moth DNA ZAL17, 3, 73, 76 and 75. The mix contained 105Microlitres of H2O,
15Microlitres of PCR Buffer, 9Microlitres of MgCl2, 12Microlitress
of dNTP, 3Microlitres of Primer and .9Microlitres of T-aq Polymerase.
By then, I had to leave, so we placed the mix with
my name on it (yeii!) in the fridge until next Thursday when I go back.
It was great to hear you talk about the things you will be doing this year. You said that so far, you've primarily only worked on theoretical things, but you were able to answer all of our questions perfectly. I had asked you to explain "bar coding" a little bit more and you did great. I had never heard of it before, so it was cool to hear about other techniques that scientists use when working with DNA. I can see the importance of bar coding, which is that you must check that the nucleotides correctly complimentary base paired with the other strand of DNA because otherwise, they DNA becomes "junk DNA." I had never really thought about checking to make sure nucleotides were correctly complimentary base pairing before, I just assumed that it worked correctly. So thank you for explaining it to me! If you have any more questions for me, I'd be happy to answer them!
ReplyDelete--Kara
Nice comments, Kara!
DeleteThanks Kara, Am glad I was able to help you out.
ReplyDeleteJosephine, great blog post. You include lots of detail and you tie it all together nicely. You must have been taking notes in the lab!
ReplyDeleteI look forward to learning what you discover.
Thank you; and yes, I have been taking plenty of notes in the lab.
DeleteTo remember the names of organisms or measurements. Look out for two new blogs about my experience in the lab. Very very exciting, I could stay there all day.
Hi, Josephine! (I feel weird calling you by your actual name...)
ReplyDeleteIt was really great to have you in my group; I was really impressed by all the questions you had for me! It means a lot to me to know that you read my work thoroughly!
Your blog is very in depth; I really like the details. It was very interesting to hear your thoughts on your work so far; even though you haven't started working with actual organisms yet, you said you had been doing stuff with moss, which seems pretty awesome, too!
I look forward to hearing more from you; feel free to ask me any more questions. I'd like to hear: what do you anticipate most from your research?
saria
Hey Saria,
DeleteI loved your blog, it was very detailed and I felt like I could duplicate the research using just the details you provided. Thank you for reading my blog, much appreciated. As for your question, what I anticipate most in the research is to just get positive results on the experiments that I am now setting up. (Look out for new blogs about actual work that I am now doing).
Interesting comments, Saria!
DeleteHi, at first even though I had hard time understanding your blog, for PCR and other DNA related works were really new to me, I became more informed of what you have been doing while I was listening to your answers for questions that Kara and Saria asked. In addition, I was really impressed with the details that you provided in your blog posts. I thought I should include more information in my blog posts.
ReplyDelete