PCR Protocol

On Wednesday (Dec. 12, 2012), I did my safety training so I can get my own access ID to building. The training was online-based and pretty simple, and I ended up spending about 2 hours on it. I didn't get my ID right away because the person in charge was off that day. After my training, I went to Dr. Koffas, but since there was only about 40 min left, he didn't assign any project to me. At the last 10 min, he introduced me to his another post-doc, Eun Ji, whom I will be working with next week. It was such a coincidence because I had met her in the lab when I was with Namita the other day. She was very outgoing and nice. I also met few of Dr. Koffas' research group members too because they were going to have a group meeting. However, because I was running out of time, I couldn't join them. Nonetheless, I hope after I have gone to my internship for a few times, I can join several group meetings as Ms. Mroczka told me those group meetings are the most interesting part of a research. Anyways, although I didn't do much on that day, I was looking forward to working with Eun Ji and do some fun experiments:)

I would also like to briefly explain the PCR protocol, in which I read about last week. PCR (Polymerase Chain Reaction) is a common used technique to amplify a specific piece of DNA (to clone a strain of DNA). PCR involves in 3 processes, each requires a specific temperature:

After putting the prepared solution into a Thermal cycler...

1) Denaturing: Under 95℃, the double helix DNA was separated into 2 single strands.

2) Annealing: The temperature quickly cools down to 55-65℃, attaching two primers (forward primer and backward primer) to the end of each strand of DNA. A primers is a strand of nucleic acid that serves as a starting point for DNA synthesis and it is required because the enzymes that catalyze DNA replication, DNA polymerases, can only add new nucleotides to an existing strand of DNA.

3) Elongation: The temperature is then  increased to 72℃, catalyzing the polymerase synthesis of DNA.

Each 95℃-60℃-72℃ counts as a cycle, and the amplification usually runs about 30 cycles. By the end of the whole process, an single piece of DNA can be amplified up to a billion strands within just a few hours.

       Retrieved from Pray, L. (2008) The biotechnology revolution: 

PCR and the use of reverse transcriptase to 

clone expressed genes. Nature Education 1(1)

In this lab, we amplify the DNA that coded for proteins required by bacteria to synthesize NP. Then, we would connect the strand of DNA into a plasmid, a designed DNA in double-circle shape.

In the end, we insert the plasmid into the bacteria. In order to keep the foreign DNA inside bacteria, the scientists also include the DNA responsible for antibiotic-resistance in the plasmid and raise the bacteria in antibiotic environment, so the bacteria would need to keep the plamid in order to survive. (I think this was really clever!)