Friday, May 13, 2016
Oh yeah I know how to…I mean I thought I knew...
The day that I heard we were doing mini preps for the first time in 20.109, I suddenly felt pretty confident. “Hey that’s something I’ve done a million times in my UROP!” I even assured my lab partner, who had not performed mini preps before, that she didn’t need to worry about there being any issues with the protocol since I pretty much knew the steps by heart.
Now nothing actually went wrong with the mini-preps that day. However, it was in the prelab prior to performing the purification that I realized I how little I actually knew about mini-preps.
I may have known what buffers I had to use, when I had to use them, and how much I needed to add of each kind. But I had never gone out of my way to understand what each buffer was actually doing. Furthermore, I didn’t even know how those nifty columns actually purified DNA. Despite that initial deflating feeling which arose when I realized I couldn’t answer most of the questions about each buffer, by the end of the prelab I felt like I had been enlightened by Leslie. I no longer was merely adding Qiagen’s P2 buffer after P1 because the protocol said to do so. I was now adding P1 (which is not just some magical chemical but I now know it should have Tris, EDTA , and RNase) in order to resuspend the pellet, weaken the cell membranes of the cells, and to eliminate undesired RNA. I was then adding P2, an alkaline solution containing SDS and NaOH, in order to lyse the cells. That NaOH is able to denature the DNA. But why add SDS now? Ahhh, since later when we add N3 in order to neutralize the alkaline solution, SDS will precipitate out of the solution in the neutral pH, carrying with it the dissolved proteins and lipids that we want to get rid of.
This is an example of one of the aspects of 20.109 that I truly appreciated the most. It was extremely useful to be able to learn new protocols while gaining an understanding of what you were actually doing along the way. Even when you may have thought that you knew a protocol very well from your previous experiences, this class also gave you an opportunity to brush up on or even learn for the first time why things worked out the way that they did. This information is of course crucial for troubleshooting if anything funky appears to happen during lab. Furthermore, this experience I just shared was a wakeup call, which made me realize that it is never a good idea to satisfy yourself with simply memorizing steps in a protocol. Many robots out there can do that just as well. Taking a few extra moments to look up the chemicals you are using before going to class or your UROP, and asking questions when you are in doubt will definitely pay off on the long run.