For the Plant DNA extraction lab a modified Alexander et al. tube protocol was followed. Filtered tips were used throughout the entire lab. Form the distributed samples, each person was assigned three different plant samples. Along with the samples 2.0 mL tubes were distributed and each person labeled three with an individual code for each one of their samples. The codes and the sample it referred to were recorded on a table sheet. A sterile 3.2 mm stainless steel bead was placed in each tube. I had three samples of mimulus cardinalis. One was collected from Mt. Diablo and the other two samples did not have locations written on them. A small amount of each leaf sample was placed in their designated test tube. In between the cutting and distributing of the samples, the tweezers were wiped down with a paper towel to avoid cross contamination. The tubes were sealed and placed in a modified reciprocating saw rack. It was attached to the saw and set to a speed of 3 for 40s. After 40 seconds, the tube were placed in the centrifuge for 15 seconds at a fast speed to remove dust plant dust from the lid. The metal bead was removed.

Next, 434μL of preheated buffer were pipetted into each tube. The tubes of mixture were placed on a water bath in a 65°C incubator for 10 min. During the ten minute period the tubes were inverted every 3 minutes. A timer was set to invert the tubes at the appropriate time along with removing the tube after 10 minutes. Then, 130 μL of #M pH 4.7 potassium acetate was inserted in each tube using a pipet. The sample tube were then moved on to ice and left untouched for 5 minutes. Following this step, the tubes were placed in the centrifuge at maximum speed for 20 minutes. My desk partner and I balanced the centrifuge with our six samples and made sure the lid was on before starting the machine.

New 1.5 mL tubes were collected and labeled with the code created at the start of the protocol. The supernatant collected in the tubes with the sample was transferred to the three new labeled tubes. Next 1.5 volumes of binding buffer was distributed to each tube, 600 μL. Three Epoch spin column tubes were gathered and given a code on of the plant sample codes. Using a pipet, 650 μL of mixture of each sample was placed in its designated Epoch spin column tube and then set in the centrifuge for 10 minutes. After, the tube was removed and the waste was discarded. The process was repeated with the remaining 650μL of mixture.

Once the second round of waste was discarded, the DNA was washed by adding 500μL of 70% EtOH to the column and set in the centrifuge for 8 minutes to allow the liquid to run through. The liquid which passed though was also discarded. The process of cleaning the DNA was repeated once more. Finally, three more 1.5mL tubes were collected and given an individual code. The tree tubes replaced the collection tubes, so the columns with the DNA ended up in the new sterile tubes. With a pipet, 100 μL of preheated pure sterile H2O was dispensed into each tube. Before placing the tubes in the centrifuge, they were placed on the rack to sit for 5 minutes. Once the 2 minute run of the centrifuge were completed tubes with the eluted DNA were capped and stored while the column was discarded.  

Image: Plant Tissue DNA Extrication