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.
