Daily Logs

6/20-6/24 6/20/11 Today, we conducted a western blot on AML12, with conditions: -Serum, +Serum, +SiD1, +SiCn, +SiCDK2. We made new transfer buffers and running buffers to test and see if the western blots that did not work last week were due to this. We let the westerns to transfer onto the membrane overnight in the freezer. We also helped Eric clean and sanitize containers and graduated cylinders with the autoclave. We plated cells by first removing old media, adding 1X PBS to clean the cell, using trypsin to disrupt the cells. Then, we evenly separated the cells into four plates and incubated them. Eric also sat down with us to discuss our project, and how we should go about proving our hypothesis. To find if cyclin D1 binds to PPar-alpha, we will be conducting western blots and immuno-precipitation. To test if cyclin D1 inhibits the response of PPar-alpha to localize into the cyptoplasm, we will use PCR. To test if cyclin D1 affects the transcription activity of PPAR-alpha, we will conduct promoter luciferous assays.

6/21/11 Today, we washed our western blots and probed for antibodies PPAR-alpha and cyclin D1. We also made new mixtures of 2.5% nonfat dry milk to prime our membranes. Afterwards, Anna and I worked on our research plan together, but quickly found out that it would be easier to complete separate sections on our own and then combine it together. For lunch, we went out to Chipotle and met up with the rest of the science research members, and also went over the grammar quiz. When we got back, we worked more on our research plan in tandem with keeping up with the washes and the secondary antibody for the western blots. We probed cyclin D1 on the machine, but discovered that the western blot once again did not

work. There were no clear lines indicating protein, and the streaks were too muddied to be identified clearly.

6/22/11 In the morning, Anna and I probed for PPar-alpha on our western blot, but it did not work. We also attempted to probe the blot with a different cyclin D1 antibody and secondary antibody, but it did not turn out either. Eric will be working with the two of us tomorrow and Friday to help us figure out what we are doing wrong. We also isolated RNA by adding chloroform, isopropyl alcohol, ethanol, and SDS solution, centrifuging the tubes between each and removing the supernatant. In the afternoon, we looked through the recipe box of how to make certain buffer and solutions used in our experiments as well as the materials listed in each experiment s protocol to determine whether or not they were hazardous, and print out MSDS sheets for the ones that are. We finished our research paper, which is good because we will be doing a lot of bench work on Thursday and Friday and will probably not have a chance to work on them further.

6/23/11 Today was a busy day in the lab today. When we first arrived, Eric helped supervise us as we re-did our AML-12 westerns from Monday that did not work. We also took more detailed notes from him as he explained what he did when he loaded his westerns. While the gels were running, we took the RNA from yesterday s experiment and performed the protocol to make cDNA. We began first by removing possible DNA left over from yesterday by mixing the RNA solution with a DNAse enzyme and buffers. To get the exact concentration of how much dH20 and condition to put in, we used an spectrophotometer to determine how much RNA was in each solution. This way, all the samples would get an equal amount. Then, we took the pure RNA and added the RNAsin inhibitor, buffers, MgCl, dNTP, and ddH20 to get cDNA. Afterwards, we prepped our membranes for the westerns by placing them in 100% methanol for ten seconds, dH20 for five minutes, and transfer buffer for 15 minutes. We compiled the western blots that did not work from the past two days into the computer to make a PowerPoint.

6/24/11 Today, we took out our transfers from the gel to the membrane. Unfortunately, the clamp was not tight enough, so not all the ladder transferred. This indicates that there may still be potential protein left on the gel. We threw out the clamps and Eric placed orders for new ones to be shipped in next week. Next, we made master mixes of each of the target genes for PPAR-alpha we had in stock in the lab CPT1A, ACSL1, ACOX1, Cd36, Ucp2 and added the reverse and forward primers of each. Next, we loaded the PCR well (which contained 96 wells) with 5 L of the RNA sample from yesterday and added each condition to each RNA sample. We did this using a hand pipette for the smaller measurement and an automatic dispenser for measurements larger than 10 L. We put a plastic film over the 96 wells and centrifuged it at 500 RPM. Then, we put the samples into a machine, where it collected data from a fluorescent reporter probe the quantitative measurements of gene

transcription. RT-PCR was also used to determine the genetic expression of a particular gene changes over time in response to the target genes of PPAR-alpha.