Wednesday, February 29, 2012
Cancelled :(
Today, February 29, 2012, I will not be able to go to my internship because of the snow.
Monday, February 20, 2012
Can't Go
Unfortunately, on Wednesday, February 22, 2012, I will be unable to go to my science internship to do a change in the Emma schedule.
Thursday, February 16, 2012
Just Another Day in the Lab
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!
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!
Wednesday, February 8, 2012
Feb 8, 2012 More Gels!!
Today as I was walking to Dr. Cryan's office, Dr. Cryan was coming towards me and in passing said that he was just called into a meeting, so he couldn't work with me today. He brought me into the lab and introduced me to Sam and Julie. Sam was talking about computer software when I came in. They explained some things about how whenever a genome for a species is completed, it gets entered into this world wide data base that anyone can view. Then, Sam went off to do his work, and Julie worked with me for the day. We ended up making two gels. The first time, I did the same things that I've been doing, making gels, inserting the DNA with a pipette, and then running gel electrophoresis. Unfortunately, nothing showed up. There was however some thing shown in the picture. Julie said it was probably excess primers that were added in the PCR. Just in case, she said we should rerun the gels and see if the same thing happens. After making a second gel, we saw that nothing appeared. This means that yet again, something was wrong in the mix. After the second gel was done, Dr. Cryan came back from his meeting. He said that next time, since we wouldn't have time today, we should use someone else's kit, someone else's station and try it again. I guess a different person tried to run it and got good results. Then we went to his instructions and compared them to mine. He had added more magnesium chloride and doubled the amount of primers. Both Julie and Dr. Cryan said that they were important changes and could very likely be the reason that my gels haven't been working properly. Next week I will start all over from using the PCR with the other person's instructions, kit, and lab station.
It was nice working with Julie today. I was able to compare different techniques and processes that the two scientists do to accomplish the same tasks. For example, Julie uses a "cross linker" to clean some of the trays before we use them. It looks like a microwave. You just place the tray into the machine, and it uses UV light to clean potential excess DNA off the tray. Dr. Cryan just uses water to clean the tray out.
All in all, I am crossing my fingers that next week's test works because we are running out of ideas. I am trying not to be discouraged, but I guess we'll have to wait and see!
It was nice working with Julie today. I was able to compare different techniques and processes that the two scientists do to accomplish the same tasks. For example, Julie uses a "cross linker" to clean some of the trays before we use them. It looks like a microwave. You just place the tray into the machine, and it uses UV light to clean potential excess DNA off the tray. Dr. Cryan just uses water to clean the tray out.
All in all, I am crossing my fingers that next week's test works because we are running out of ideas. I am trying not to be discouraged, but I guess we'll have to wait and see!
Wednesday, February 1, 2012
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