Today was a really good day and I'm excited to tell about it. Flashback to yesterday, right as Tom and I were about to start my first experiment, Doctor Willey realized that they had to do something very important for this article they are about to publish. We stopped and Tom did his thing. Flashforward to today, for the whole morning, Tom and I set up proper dilatations of DNA so that we could titrate properly (essentially we are just trying to find how many of the unknown molecules we have based upon a set ratio of fluorescence). Then, Tom walked through the experiment, doing all of the pipetting (which is a pain) and explaining why he was doing each step and telling little helpful tidbits of lab procedure. He then had me do the exact same thing back to him. Luckily, I pick up on things quickly, but just the pipetting caused my muscles to shake and nearly spasm. It looked so much easier when he did it! But, he did say that I did a very good job (even better for my first time... EVER). We put the samples we had made up into the PCR machine. We then went to lunch. Yes, I know. Very anticlimactic.
We then came back (I did faster than he because he got stuck at Fricker's) and ran the amplified samples through a fancy and expensive (worth over $30000, I didn't even think about touching it) machine that performed electrophoresis to see how long the strands are. Just to give a frame of reference, our DNA in each cell has billions and billions of base pairs. The genes we were interested in have roughly 500. Then, based upon different dilutions, we could set up proportions to see roughly the amount of molecules from this test. We are testing to see if two different extraction methods have RNA (therefore cDNA) that are in the same proportion.
....You should be confused. I sure was, and if you have any questions, please ask!
Below are pictures of where I worked:
PCR machine:
My little comfy station: 
OK. I"m confused. Why do you care how long the strands are? What does different dilutions mean?
ReplyDeleteThe strands' varying lengths mean that they will get pulled differently in electrophoresis (click on the hyperlink for more info). Since the longer ones will pass a certain point after the shorter ones will, we can calculate their base pair amounts based upon how long they take. We do this to separate the molecules to see how fluorescent each length of molecules was when they passed over a set light source. We then can set up a proportion
ReplyDeleteFluorescence of known amount of molecules Number of known molecules
----————----————----————----—— = ----————----————
Fluorescence of unknown amount molecules Number of unknown molecules
Thus, we can determine the number of unknown molecules.