10/17/18 Lab Write Up #8
This lab focused on extracting DNA from plants from different locations all falling under the species of Mimulus cardinals or Mimulus guttatus. Only one of my three sample IDs were labeled with location and name which was JP1321 (A) Mimulus guttatus from Mill Creek Mono North. The other two samples had the IDs JP1297 (B) and JP1300 (C). The tissue samples from these plants were left in silica beads to soak up any moisture to improve the DNA recovery from the sample. Due to the difficulty of extracting DNA from plants caused by the cellular structure and cell wall, I used 3 small metal balls for each sample to break down the tissue into a powdery poultice. The epi tubes that contained the samples and the 3 balls were then shaken extremely fast with a modified power tool for 40 seconds. Carter volunteered to use the power tool to break up my samples. After the samples were in a powder I used the protocol handed out for DNA extraction. This included adding a heated reagent followed by 10 minutes in the water bath during which the samples had to be inverted every 3 minutes to ensure proper exposure. I then used the centrifuge to spin down my samples to get rid of any dry solids and/or dust. Following this, acetate was supposed to be added. I had a small issue here because I was handed an uncapped falcon tube and was told it was the acetate, only to find out after that it was truly ethanol. I had to restart the protocol from the beginning, but was luckily able to catch up with the rest of the class during lecture. After reaching the acetate step once again I spun down my samples for 20 minutes. I then added the supernatant with another reagent at a 1:1.5 ratio. My samples all had 400 microliters of supernatant so I used 600 microliters of reagent. This was then pipetted in a two step process (due to large volume size) into individual column tubes to start the DNA collection process. After the all the supernatant had run through the column assigned to that sample, the columns were then washed with ethanol. Once the ethanol washes were completed, it was time to get the DNA from the column into the fresh correctly labeled epi tube. To do this I washed with warm water, let it sit for 2 minutes and followed that by centrifuging the columns with the collection epi tubes. The result was collected DNA in epi tubes that will be amplified and further analyzed for proper collection.