October 22, 2019
In this lab, we were given the sample of the Mimulus guttatus leaf that we had picked in the previous field trip that we had taken. We also were given two more samples of leaves that Alec had stored for us. When I was given the leaves, they were dried up in the tubes full of silica. Before we began using the leaf samples, we labeled 3 2.0 mL tubes with our samples codes. The first sample was labeled ‘RDRK 001’ the second was ‘EB’ and the third sample was labeled ‘SHOR 005.’ In each tube, I added three sterile 3.2-mm stainless steel beads to each tube. I added a small amount of leaf tissue to each tube using clean tweezers between each sample to avoid cross contamination.
I capped my tubes and loaded them within a tube rack into the modified reciprocating saw rack and mounted the rack to the saw. Professor Paul made sure that the blade was completely secured and locked into the saw before plugging in the saw. Professor Paul put safety goggles on and turned on the reciprocating saw on speed 3 for 40 seconds.
We briefly spun down the tubes in the centrifuge for 15-20 seconds at fast speed to pull plant dust down from the lids. After the plant dust was pulled down, I added 434 μL preheated grind buffer to each tube. We incubated the buffer grindate at 65°C for 10 minutes in a hot water bath. We made sure to mix the tubes by inverting them every 3 minutes. After incubation, we took the tubes out and added 130 μL of 3M pH 4.7 potassium acetate into each tube. The tubes were inverted several times and incubated on ice for 5 minutes. After ice incubation, we took the tubes out and placed them in a centrifuge at maximum force for 20 minutes making sure that the centrifuge was balanced.
We labeled new 1.5 mL tubes with the sample ID. I transferred the supernatant to these sterile 1.5 mL microcentrifuge tubes avoiding the transfer of any precipitate. I added 1.5 volumes of binding buffer.
We applied 650 μL of the mixture from the previous step to the Epoch spin column tubes and centrifuged the tubes for 10 minutes until all the liquid has passed through. The centrifuge was placed at 15,000 rpm and the flow-through was placed in a hazardous waste container once the centrifuge was complete. This step was repeated once more using the remaining volume from the previous step.
We washed the DNA bound to silica membrane by adding 500 μL of 70% EtOH to the column and centrifuging it at 15,000 rpm until all the liquid passed through to the collection tube. This process was done in 8 minutes and the flow-through was discarded. Repeat this step once more.
After discarding the flow-through from the last step, we centrifuged the columns at 15,000 rpm for an additional 5 minutes to make sure that any residual ethanol was removed. We were instructed to discard the collection tubes and place the columns in sterile 1.5 mL microcentrifuge tubes and add 100 μL of pure H2O. However, I made the error of placing the centrifuged columns into the tubes that contained the binding buffer and place 100 μL of H2O. When I caught my mistake, Professor Paul advised me to transfer the columns into clean 1.5 mL tubes and add an additional 100 μL of 65°C H2O to each tube and let it sit for 5 minutes. After this was done, I put the tubes in the centrifuge for 2 minutes at 15,000 rpm to elute the DNA. The new tubes were labeled ‘EB 1’ ‘EB 2’ and ‘EB 3’. I drew a star on the tubes that the columns were initially put into so that we may use them in the future if we find that no DNA was extracted into the new tubes.
