Today was an interesting day.
In the morning, I did some PCR with ACTB (the reference molecule for nearly all of the tests in Dr. Willey's lab) and CEBPG (the gene that I'm focusing on). I felt like I did a better job of running the test and I hoped that my data would turn out well. I waited for the tubes to go through PCR, then I loaded up the set into the electrophoresis chip. Everything was going according to plan, and I left for lunch so that I could look at results when I got back.
When I got back, I saw a blank screen. Normally, when I screw something up, the reading swill just be odd and off by a considerable margin. Not this time. Literally, I got no data. Naturally, I started getting worried; I thought I had broken the machine, which easily costs more than few month's tuition for MV. I ran to Erin (#2 in Doctor Willey's Lab) and asked her what happened.
Well, it turned out that the chip never ran. Apparently, every once in a while, the laser that is used for the electrophoresis doesn't get focused properly, so the entire process stops. Slightly annoyed that the machine didn't tell me so immediately, I needed only to load another chip because I had been wise enough not to throw my samples away (go me!)
Eventually, I got my data. Despite my struggles of getting back into the PCR groove, 3 of 8 samples were what they were supposed to be. 4 of them were wrong because we realized that my primer for the CEBPG (a necessary part of the PCR) wasn't working right, so it should work next time. However, 1 of the 8 was wrong and it's a good thing that it's wrong. One of the more interesting parts about the methodology of these tests is that I'm measuring the molecules relative to the known number of molecules that we insert. Now I have to balance these and get them into a ratio between 10:1 and 1:10 to make accurate conclusions. This means that I have to run more tests, which I'm excited for.
Anyways, if you saw Mr. Boehm's comments, you know I have some explaining to do. Check tomorrow to see my updated SMART goals and other new things for my Winterim.
Sounds like a frustrating day! Now that you know the PCR occasionally malfunctions will you do anything differently? Good thing you saved your samples.
ReplyDeleteSpeaking of samples, it isn't clear to me why getting 1 out of 8 wrong is a good thing? Please clarify.
The PCR itself doesn't malfunction, but the machine reading the results does. Since this happened, I wait until the test starts normally before walking away.
ReplyDeleteThe sample wasn't truly wrong, but it wasn't as right as it should be. Normally, we like to have a ratio between 1:10 or 10:1, and for this sample, the ratio was roughly 1:20. Thus, the data wasn't usable. This isn't my or the machine's fault. I just hadn't known that this sample of cDNA needed a different test setup to be read accurately, something I've corrected.