December+2011

=toc December 2011=

Week December 5
We met today and Francis and I discussed the paper he gave me the following Wednesday about JAK-STAT pathways. A link to the article can be found below: [] We discussed how the SOCS is turned on and off within the cell and how the STAT 3 dimerizes and can enter the nucleus. I learned that a kinase is a phosphorate and a phosphotase de-phosphorates within the cell. I also learned that STAT 3 is a transcription factor, which means it can turn on and off multiple genes in the nucleus. I then drew a picture of what the pathway would look like: media type="custom" key="11722668" These were some pictures in the article that helped me to imagine what was going on and how the different elements connect together in the pathway: media type="custom" key="11722742" This figure show the different JAK combinations mostly used by cytokine receptors, which allow things in and out of the cell media type="custom" key="11722792" The description below the figure explains the picture media type="custom" key="11722820" Three classes of genes activated by STAT 3 are shown
 * December 5, 2011**
 * December 7, 2011**

We also reviewed where diferent processes that happen inside the cell: media type="custom" key="11722374"

Week of the 12
I set up a PCR to sit at 37 degrees until Wednesday in the incubator used for PCR. We used xcm1 which should cut the GFP at about 100b and we should also get linearized vector once we run the gel Wednesday we should see only two bands on our gel. We worked with Francis and ran a gel to show us our two bands that we hoped for to show that the PCR worked. We then wanted to extract and dissolve the bands to get the GFP and the vector so that we can hopefully religate them again. But after dissolving and nanodropping our results, we saw that it had not worked according to specking them because we did not see a peak at 280 as we had hoped but instead saw one at 230. And below is how we dissolved our gel pieces.
 * December 12, 2011**
 * December 14, 2011**

Gel Extraction (QIAquick)

 * 1) Excise DNA fragment from agarose gel with clean, sharp scalpel.
 * 2) Weigh gel slice in colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100mg ~ 100µl)
 * 3) Incubate @ 50C for 10 min.
 * SOLUBILIZE AGAROSE COMPLETELY
 * To help dissolve gel, vortex tube every 2-3 min during incubation.
 * 1) After the gel has dissolved, check that the color of the mixture is yellow. If not, add 10µl 3M NaOAc. Or just add the NaOAc anyway.
 * 2) Add 1 gel volume of isopropanol to the sample and mix
 * Do only if 4kb
 * 1) Place spin column in 2mL collection tube.
 * 2) To bind DNA, apply sample to column. Centrifuge 1 min.
 * 3) Discard flowthrough and place column back in same collection tube.
 * 4) Recommended: Add 0.5mL of Buffer QG to column and centrifuge for 1 min.
 * 5) To wash, add 0.75mL of Buffer PE to column and centrifuge for 1 min.
 * 6) Discard flowthrough and centrifuge for an additional 1 min @ 17900 x g (13000rpm).
 * 7) Place column into a clean 1.5mL microcentrifuge tube.
 * 8) To elute DNA, add 30µL Buffer EB to center of membrane, let column stand for 1 min, then centrifuge for 1 min ( we used 20 instead to hopefully get more concentrated DNA) []