From 2007.igem.org

Nnguyen 15:40, 26 June 2007 (EDT) Today (June 25,2007) I came in and I started my tutorial on internal sites because I missed a week of work last week. I completed the tutorial, and I was given my task for the week. Chris gave me a template to work on. The gene is called Streptomyces chrysomallus. There are four genes of interest in the template, and I had to make oligos for these genes. I had to construct two oligos for each gene, a forward primer and a reverse primer. In order to construct the oligos, I used the enzymes BglII and XhoI. BglII was used for the forward primer and XhoI was used for the reverse primer. In 2 out 4 genes, there were internal sites which meant that I had to make additional oligos.For gene ThpA, EcoRI was within it and so I had to make a primer that would remove it. The sequence for EcoRI was gaattc, and I changed it to gagttc. gag and gaa both code for the same amino acid which is glutamic acid. XhoI was within gene ThpB, and again I had to make a primver that would remove the restriction enzyme. The sequence for XhoI is ctcgag, and I changed it to cttgag. ctc and ctt both code for the same amino acid which is leucine. In order to remove XhoI from ThpB, I construct a forward primer and a reverse primer. The reverse primer is the complimentary side of the forward primer. Compared to the oligos that I constructed for the beginning and end of each gene, the primers used to remove XhoI do not have restriction enzymes on them. Compared to ThpB, ThpA only has 2 primers and not 4 primers because the EcoRI was very close to the beginning of the gene. Therefore, I incorporated EcoRI into the forward primer for the gene ThpA. In the end, I ended up with 10 oligos.

At the end of the day, Sam showed the high school students how to pour agar into plates, and we left the plates on the counter throughout the night.

Nnguyen 15:40, 26 June 2007 (EDT) I came into work today and helped the other high school students a little bit with their project while I was waiting for my oligos to arrive. They have not arrived yet; however, in the meantime, Sam and Arthur showed us how to make LB Agar and LB Broth. We poured 500mL of water into each glass bottle. For LB Agar, we added 20g of agar powder, and for LB Broth, we added 12.5g of broth powder. After adding the right amount into the 500mL of water, we shook the bottle until the powder dissolved. After that, we put all the glass bottles in a tray, untwisted the cap so that when they're put in the machine, they wouldn't break due to high pressure, and we added water and tape the caps. After we finished with the preparations, we put the tray into the autoclave machine for 25 minutes; however, it is better to let it sit in there for awhile to let it cool down.