Lab 8 Entry
10/17/18 Wednesday. Wet lab: population Genetics project- DNA extraction (plant) and Gel Electrophoresis 1
In the beginning of the lab, I received a sheet of paper with the title Plant Tissue DNA Extraction and table consisted with Sample ID, Location and Species. Then I received three samples from Prof. Paul and those were JP 1319 mill creek mono north M.guttatus JP 1303 and JP 1296.
Modified Alexander et al. tube protocol for DNA extraction
Note: Always use filtered tips for all steps in this protocol.
First, I labeled my given 3 2.0mL tubes with my sample ID (JP 1319, JP 1303 and JP 1296). Then I added three sterile 3.2mm stainless steel beads to each tube. Next, I took my tweezers and took out my sample leaf tissue, the size of my thumb nail, and put it into the given tubes with the label. I cleaned my tweezers with ethanol every time I took out different sample leaf tissue. As a group, Peter loaded the tubes within a tube rack into the modified reciprocating saw rack and mount the rack into the saw. He pulled the trigger and let the machine shake for 40 seconds. This process is to mush the leaf tissue and make it like a powder like. After 40 seconds, as a group again, we put each of our samples and briefly spin down the tubes in the centrifuge for about 30 seconds at the maximum speed to pull plant dust down from the lids. I then took out my sample tubes and added 434 micro liters of preheated grind buffer to each tube. After that, I incubated buffered grindate at 65 degrees Celsius for 10 minutes in water bath, mixing the tubes by inversion every 3 minutes. After 10 minutes, I took out my sample and added 130 micro liters 3M pH 4.7 potassium acetate, invert tubes several times and incubate on ice for 5 minutes. I waited for the entire class to start centrifuging at the same times. After everyone was done, as a group, we put our samples in the centrifuge and spun it at maximum force for 20 minutes. While it was getting centrifuged, I labeled new 1.5mL tubes with the sample ID (top and side). I also labeled Epochspin column tubes (blue on the bottom, white with filter on the top). I will be adding supernatant and it will capture DNA and filter out the waste. After it was centrifuged, I transferred my supernatant (on the top) to these sterile 1.mL microcentrifuge tubes. After transferring my supernatant, I added 450 micro liters of binding buffer into JP 1319 and JP 1303 and added 600 micro liters of binding buffer into JP 1296. I then added 650 micro liters of each mixtures to each of Epoch spin column tubes (correctly labeled ones) and centrifuged it for 10 minutes. After it was done, I dispose the buffer (in the blue tube) into the hazardous waste bottle and repeated the same thing again. After centrifuging all my mixtures, I added 500 micro liters of 70% EtOH to the column and centrifuged for 8 minutes and discarded the flow-through. EtOH is to wash the DNA bound to the silica membrane. I repeated the same thing again and at the third time I centrifuged for 5 minutes without adding anything in the column to dry out any residual ethanol. I labeled another 1.5 mL tubes with sample ID and date, which will hold my final extracted DNA. After ethanol was all dried up, I threw out the blue bottom and switched with 1.5 mL tubes. I then added 100 micro liters of preheated (65 Celsius) pure sterile H20 to each tube, let it stand for 5 minutes and centrifuged for 2 minutes to elute the DNA. After it was centrifuged, I threw away the column (top), capped the bottom and put it into the ice box.