Yesterday, February 16, 2012, I went to the NYSM again. Like I said in my last post, my mentor decided to have me start all over by making the mix for the PCR but we increased the amount of Magnesium Chloride and primers. Instead of making 2 mixes for the PCR, I made 5. We worked with DNA sections 18S and 28S. I made the gels as I always do and then put them into the PCR machine. I was working with Dr. Cryan again this week. After we put the mixes into the PCR machine, Dr. Cryan took me into his office so we could look at a computer data base for DNA. He explained the process of what happens to the DNA after we do the gel electrophoresis. Since none of my gels have come out perfectly, we haven't gotten to the stage of sending off our data, but hopefully this gel will turn out right and then I can send off my mixes for real. Here's the process:
After doing gel electrophoresis, you should see a lot of glowing bands which signifies that the DNA is good, the components of the mix were done right, etc.
Then we ship off the mixes to a center that puts the mixes through another PCR to amplify and make more copies. The only difference is that in that PCR, they include some primers that are fluorescently tagged. In our PCR, we include regular A,G,C, and Ts. This other PCR has some of the regular ones but also has special A,G,C,Ts.
That place then ships the amplified mixes to another place that has a machine that is sort of like gel electrophoresis but more advanced. The gel isn't made of agar, but something different, and instead of being 6 inches long, it can be 5-6 meters long. The mixes are put on the plate and electrical currents are run through it. The special A, G, C, and Ts do two things. One, they have a stopper on the end, so when the PCR was amplifying, the DNA could only replicate until it attached a special primer that stopped it. So you would end up with
A'
AG'
AGT'
AGTA'
AGTAC'
and so forth.
The length of the chain made will determine how far down the plate the chain can travel. At the end of the plate, a laser is shot through the special primer. This causes it to fluoresce. A computer then takes a snap shot and records the color that the nucleotide glows. There is a universal code for each color corresponding to a nucleotide. I may be incorrect but I believe the the colors are
A -- green
G -- red
C -- blue
T -- black
After all of the nucleotides have been recorded by color, the computer matches the color to the letter. This data is then published and we can look at the data and move it around.
I hope the gels work next time so I can start to work on this process!
I totally understand when you have to start all over again! One time we had to fix all of our tools because they had fallen and spilled all over the floor. Even though it didn't completely mess up my results, it was another unexpected moment in the lab. I give you many kudos for having to start your mixing and experiment again. Seeing that you have had to restart a couple of your experiments, I was just wondering if you have any extra precautions. Maybe I can be more careful, too!
ReplyDeleteThoughtful comment, Caroline.
DeleteGood blog, Kara. I like the amount of information that you included, as well as your optimistic attitude. You do not let the missteps get you down, which, as Caroline commented, is a key part of the scientific process. Keep it up!!
ReplyDeletePCR is definitely one of the procedures that you have to be extremely careful with! One mistake and the results won't turn out properly! I remember learning DNA strands in biology class last year. I only read about special primers; but it is quite fascinating that you can differentiate them through gel electrophoresis! Since the things you are working with are so minuscule, how can you tell the difference between a regular and special nucleic acids? I know that you can differentiate A C G T by the lengths, but I was wondering how is A' different from A?
ReplyDelete