IC, UC, 3C
Welcome Fish Friends,
In the PC Summer Research Labs, science based offenses are considered especially heinous. In the Weicksel Lab, the dedicated scientists who investigate these felonies are members of an elite squad known as the Science Investigators Unit. These are our stories.
Weekly Updates:
This week begins week 5 of the 10 week journey for the Weicksel Lab summer research group.
On Monday, we began the long awaited 3C: chromosome conformation capture. The 3C protocol is 4 days long so Monday was day 1. This day involved adding chemicals and then waiting and then adding more chemicals and doing some more waiting, also known as digestion day. The embryos we froze after fixing them for 3C finally got to be this weeks stars (or should I say starfish?). Remember the buffers from last week? Well those are the first chemicals we add to our tube of embryos. Then we put it on some ice. We added another buffer, and mixed that into one tube and then put that mixture into a fresh tube. The purpose of this is so that we can get the nuclei from the embryos. After doing a lot of steps similar to this, we got use a fancy machine called the confocal microscope. Before we used the machine we stained the nuclei with DAPI, which is a fluorescent stain that will bind to areas that are high in concentration of A-T. The confocal microscope allows us to see the nuclei in our sample. We want to see how many nuclei are in the sample so that we can get an idea of how much DNA we have. When we finished with the machine we added some more chemicals and incubated those tubes all night long.
Tuesday is day 2, ligation day, of the 3C protocol. Day 2 brought about as much incubation as a person can handle. We started by adding SDS and then incubating for 20 minutes. Then we had to add ligation master mix and incubate for... 4 HOURS!!! We had a lot of down time during that so we made some fish system updates, by adding little valves to the output so that we can control the flow of water to each tank. After our 4 hours ended, we added some proteinase K and had to incubate the tubes overnight.
Wednesday began day 3 of the protocol. We can see the finish line of this protocol, only 1 more day. We pulled our tired tubes out of incubation in the morning and added some more proteinase K to wake them up. Then we let them rest in the incubator for another 2 hours. Much like the other days we then added another chemical and incubated again. After this incubation we got fancy and did a phenol chloroform extraction. Phenol chloroform extraction allows us to just get the nucleic acids out of our samples. When we do this, we add phenol and spin the sample in a centrifuge. After we take it out of the centrifuge it has two distinct layers, the top is the nucleic acids, the bottom is proteins and basically everything else. We repeat this process once with phenol and once with chloroform. Then we proceed to an ethanol precipitation, so that we can precipitate nucleic acids. This just means we want the DNA to be more like a solid so that it is easier for us to get. After this we leave it in the -20 freezer for an overnight.
On Thursday, we wrapped up with our final day of the 3C protocol. We had to continue the rest of our ethanol precipitation from yesterday. This mostly involved washing the DNA pellet with ethanol. We then took the liquid out and let the pellet dry, before re-suspending it in water and transferring it to a system of tubes similar to the ones we used for our gel extraction. When in these extra special and fancy tubes, we had to centrifuge it again, and then took our sample over to the nano drop to discover how much DNA was in there. I would love to report that we saw a bunch of DNA, but really it was quite a tiny amount. We took our sample and put it in the q-PCR machine, to see if we could amplify a certain sequence and figure out how much of that sequence we had.
Friday, we got to see the results from our q-PCR. Even with the tiny amount of DNA we had, we managed to get some results. This is promising for the Weicksel Lab, because this is our first trial run with the 3C protocol. The lab took a lunch outing to Lasalle Bakery where we all very delicious sandwiches. We then mated some fish and collected their embryos, so that we can fixed them for 3C, so that some time soon we can do our 3C protocol again. We also had to run a gel of our q-PCR results. Sadly our data was no good, so we will try again soon.
As always thanks for swimming by! We hope you give us a swim by next week too! Any questions? Head over to our contact page.
Swim By Again Soon,
The Weicksel Lab
