Even though the chips are still in the mail, I still wanted to do a lot of work. Doctor Willey and Xiaolu want to make an abstract before an upcoming conference, so there was an abundance of work in the lab.
(The list of all the work)
My job for this week is to get the ERCC5 and ERCC4 values for cDNAs from 3 different cells lines. I set up the test and ran the PCR. So that the PCRed samples do not deteriorate, I'm going to put them into the freezer. I currently have 25 samples that are waiting in the freezer for when the chips get back. I still have 24 more samples to make.
BUT I learned a really cool new test today called Real Time PCR. Xiaolu taught me that this test has 2 steps. For the first step, she puts in the different samples in a rack with 8 wells by 12 wells. She puts the same reagents in and for 18 cycles, it goes through PCR. During these cycles, the signals that the cDNA produces get amplified. Then, the samples are diluted to 1/10, 1/100, and 1/1000. I don't know the reasoning, but then they undergo another 14 cycles of PCR. They then can determine how many molecule of the gene was produced per million ACTB (exactly the same thing I did with electrophoresis) based upon how much fluorescence is produced (it's produced by the probe finding its complementary sequence [check the wikipedia article it's kind of hard to explain]). Anyways, we get the same results, quicker and more accurately. Xiaolu and I are looking at how siCEBPG affects the production of MUC5B (another gene) in the cell lines.
Also, I talked to Xiaolu and the reason that they are looking siCEBPG knocked down cell lines is that they want to see (if there is) a mechanism involved when CEBPG regulates cell functions. I'll try to explain that more tomorrow.
Glad that you'll explain more tomorrow! Remember that your goal at Winterim Fair is to make your work comprehensible to as many people as possible-- from fellow students to experts.
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