Week 4

On Monday, we ran another gel electrostasis plate, which we would load all of our final samples in and two DNA step ladders. We did this to make sure that the samples of DNA we were getting ready to clone were good enough to use (since we will be presenting this information at the symposium). While the gel was running, we got a chance to check on our own leishmania that we started to grow last week. My leishmania grew so well, that I had to switch growing chambers and feed them some more. But before I did that, I got to look at my protozoas through a microscope and see a live beginning stage of what they actually look like. I was pretty neat. I also took a sample of the protozoa out, so that I could learn how to stain a slide and find my protozoa on the hemocetometer grid. It didn’t quite work, so I gave it another try on Tuesday. Hanna and I also worked on our slideshow presentation.
On Tuesday, we ran our final DNA on a gel plate, so that we could clone the DNA. The DNA that I was going to clone was called “Sigma.” Once we ran the plate and viewed it under the UV light and took photos, we used the UV light to find our DNA in the gel and we cut it out. It was pretty cool to see what it actually looks like face to face, instead of looking at it on the computer screen. Once we cut out our DNA, we cut it into 6 pieces and put them in very small containers. When we came back from lunch, we measured the weight of each piece of gel in each container, so that it weighed around, .400 milligrams. Afterward, we used the “PureLink Quick Gel Extraction” Kit, which purified the DNA fragments we took from our agarose gels. We did this so that when we would do our cloning on Wednesday, we would have only DNA in our clones, and not any other fragments of gel or extra stuff. Then we put it in a freezer overnight, at –20 degrees C, so that it could precipitate. This was called our PCR product. We also tried to stain another slide with our protozoa, using the “giemsa blood stain” chemical. And again, there was still not luck. So we decided on Thursday to try it again, so that our protozoa can grow some more, and we also didn’t want to use up all of our protozoa early trying to make slides, or we would have none left to watch grow. Again, Hanna and I worked some more on our slideshow presentation.
On Wednesday, we did the actual cloning process. It was very sterile and you have to be cautious and do things slowly. First, we had to prepare the DNA which we let precipitate overnight. The steps (for the PCR product) were as follows: Spin the DNA down (in the centerfuge) for 10 min., remove the soup (the liquid around the little transparent DNA pellet), let the container (with the pellet still in it) air dry for 10 min., add 8 microliters of distilled water to it, and let it sit at 65 degrees C for 10 min. Then, we were ready to do the cloning process, which only took 10 minutes. For the cloning, we: added 1 microliter of salt solution to a pre-measured PCR-4 TOPO solution. Then we added 4 microliters of the PCR product to it. Then we let that sit for 5 minutes. Next, we did the transformation. It was a long process, so in short: we added some stuff to the clone and basically transformed the DNA clones and put them on a kanomycin plate, to sit at 37 degrees C, so we could see if we cloned our DNA right. If we did our process correct, bacteria would grow on the plate, in nice round colonies. Again, Hanna and I worked some more on our slideshow presentation.
On Thursday, we saw whether or not our bacteria grew. My bacteria did grow, so I got 10 (1.5) milliliter tubes and labeled from S1 to S10 and put a colony, with 500 microliters of kanomycin, to make the colonies grow faster and better. We basically isolated them to see how (and if) they grow. I plated the rest of my clones on another kanomycin plate too. Then later on, we did the same thing we did on Monday and Tuesday with the leishmania (protozoa) and tried to make another slide. Dr. Porter-Kelley said that they should grow a lot over the weekend. Again, Hanna and I worked some more on our slideshow presentation. This week was pretty fun and relaxing. I learned a lot. I am actually pretty excited to do our presentation at the symposium, so I can explain and show what I have learned with Dr. Porter-Kelley.

Week 3

This week we began a new project dealing with microbiology under Dr. Porter-Kelley at WSSU. On Monday, we learned what we would be doing. We would be looking at different types of DNA and RNA and cloning them. Dr. Porter-Kelley works with leishmania (L. Major, is the main type) which is a genus of flagellate protozoa, several species of which cause leishmanisis; all species are indistinguishable morphologically but may be separated by their serological reactions, by their geographic distribution, by their developmental patterns in their sandfly hosts, and by their clinical manifestations of leishmaniasis. While doing work with leishmania, each of us will be growing our own leishmania and taking care of it (basically, we are breeding bacteria). We will get to see how they grow and develop over our 3 weeks with Dr. Porter-Kelley. We also were taught how to run Agarose gel Electrophoresis and we tried one of our own, just to get us in practice for when we use real DNA or RNA. I left early for a funeral, so I didn't get to see what we did to the gels after we finished using electricity on the gels after an hour.

On Tuesday, we got a wonderful break. On Wednesday, we started to grow our own leishmania. We also ran our own real Agarose gel Electrophoresis. We picked one of four types of DNA/RNA and made a 25 microliter RXN Volume with our DNA types. I used sigma in my mix. The mix was made up of: "PCR Masta Mix, 2X" (12.5 ml), "Sigma Upstream Primer" (1 ml), "Sigma Downstream Primer"(1 ml), "DWA Template" (1ml), and "H2O"(9.5 ml). I was give 4 tubes of the Masta mix and added everything to each container, 4 seperate times. I used 4 containers because each container was put through 30 cycles in the thermocycler at different temperatures, 55.2, 57.4, 64.5, and 69.8. We also made plates, which was where first, we measured out LB Agar and LB Broth and put them in two different containers and added 500 mL of distilled water to each bottle. Then we shook them up and put them in the auto-clave to be sterilized. After that, using a sterile technique, we first added 50 mg/ml of Kanomycim into the LB Agar. Then by keeping the bottle near the flame, we poured the LB Agar into petri-dishes, since we will also need those to grow bacteria. After we poured the Agar into the petri-dishes, we let them sit overnite. After we finished pouring, our gels had finished running, so we out them in the machine, and using a UV exposure light, we took pictures of them. The pictures didn't come out like they should have, so we will have to do the whole gel procedure and mixing of the chemicals over again today. This week has been pretty fun. I have enjoyed it. It has been a different change of pace working with Dr. Porter-Kelley, in a good way.

Week 2

This week flew by fast. On monday, we did a caffine extraction from tea and purified it. This would also be one of the experiments we would be teaching to the Sci-Tech program, which was for rising 8th graders, on Tuesday and Wednesday. After we did the isolation of the caffiene, we purified it using the sublimation process. Then, we went to luch and went over to WSSU to see our other mentor's lab, which we will be working in for the next 3 weeks. On tuesday, we headed over to WSSU soon as our mentor (Dr. Harp) arrived to set up for the Sci-Tech program. That day, we taught them how to male asprin. We mainly supervised them. I worked with Asia & Nzinga, Darrus & Cedric, and Joshua & Lance. Went weren't allowed to do anything for them (which is what our mentor told us to do), but I helped them out just a little. We workred with them until 3, then we came back to PTRC and did some calculations for our next experiment. On Wednesday, we went back over to WSSU to teach the kids how to extract caffiene from tea. It was funm teaching the kids in my opinion. When we were finished with the kids, we came back to PTRC and my partner and I (Darren) set our experiment, which is the Synthesis of Diphenolmethoxypiperidinol. It is a drug to try to help weeing people off of cocaine. Mixing the compound will take a few days, so luckily, we set it up correctly before we left and it stayed up over night. On Thursday (today), we came in and checked on our experiment. We ran a TLC test to see if we had any of our starting material still left in our compund we created. We do, so we are still letting it run as I type. This week was fun, but it was also pretty chill. We got to go over to WSSU and help our another science program, which was cool and we kind of got to walk around campus and see where we would be the next 3 weeks with our other mentor (Dr. Porter-Kelly). On Friday we had our GCRC sessions and worked in the computer lab. We also went to the patient simulation lab. It was quite an experience. Good or bad? Who am I to judge. It was very memorable and insightful though. I did learn a few things though.

June 20-23, 2006 - The Rest of Week 1

In a nutshell for the rest of the week, we basically got started. We were given an experiment to do, which was called the "Synthesis of p-chloro Benzhydrol". This would be the first part of our over all project, which is to create a drug that will weein people off of cocaine. First, we ran over calculations and figured out the chemical make up and how to determine the amount of the certain substances that we needed. After that, my partner (Darren) and I tried to reduce the amount of p-chloro benzophenone using sodium Borohydride. After that, the rest of the week flew by pretty fast. We ran some tests on our compound that we made to find our the purity of it. We also learned how to do a few "non-fail" experiments (which was the making of aspirin and the extraction of caffiene from tea), that we will teaching to the SciTech program, which is another science-based program, made up of 8th graders. On Friday, we had a GCRC session and went to the computer lab to work on our blogs (which i'm doing now). We also went downtown to the PTRC building located beside the one I'm usually in and went on a tour. We were basically told and shown the planned expansion of the PTRP and why having a science and math major would benifit us later on, since the park should be built by then. Overall, week 1 was alright, I enjoyed getting off early on Friday, alot!

June 19, 2006 - Day 1

Today we got the ball rolling. First day of the program. Nothing much exciting really. We (my group and I) went downtown to PTRC to meet up with our mentor (Dr. Harp), who works in the Phisiology and Pharmacology department at WSSU. We learned what we would be doing over the summer for the next 6 weeks. We are basically trying to come up with a drug to weein people off of cocaine and methanphetamine, by using a drug just as potent and controlling the amount of the drug that will enter the body.