This lab period utilized a modified version of Alexander et al. tube protocol for DNA extraction.
Materials and Methods
- 2.0 mL tubes
- Sterile 3.2 mm stainless steel beads
- Mimulus guttatus leaf tissue (preserved in silica)
- Tube rack
- Modified reciprocating saw rack
- p20 – p1000 pipets and pipet tips
- Grind buffer
- 3M pH 4.7 potassium acetate
- 1.5 mL tubes
- Binding buffer
- Epoch spin column tubes
- 70% EtOH
- Pure sterile water
I labelled 3 2.0 mL tubes with the codes RWCK 003 AJC, MAPL 005, and CATB – EVR. The first two samples were obtained by Alex, a graduate student working with Professor Paul. Three sterile 3.2 mm stainless steel beads were added to each tube and a small amount of leaf tissue from each sample was placed in their respective tubes. The tubes were placed in a rack, which then was placed in a modified reciprocating saw rack and was reciprocated on speed 3 for 40 seconds. The tubes were placed in the centrifuge for 18 seconds to pull plant dust down. 434 microliters of preheated grind buffer was added to each tube. The buffered grindate was incubated at 65 degrees Celsius for 13 minutes and 30 seconds and were mixed by inversion every three minutes. 130 microliters of 3M pH 4.7 potassium acetate was added and mixed by inversion. These were incubated on ice for 5 minutes and 5 seconds. Samples were then centrifuged at maximum force for 20 minutes. New 1.5 mL tubes were labelled with the aforementioned sample IDs and the supernatant was transferred to those tubes. Some precipitate was pipetted into sample 3. 1.5 volumes of binding buffer was added which was around 550 microliters for each of the samples. 650 microliters of the mixture was added to Epoch spin column tubes and centrifuged for 10 minutes at 15,000 rpm. The flow through was discarded in a hazardous waste container and then the process was repeated with another 650 microliters. The DNA bound to the silica membrane was washed by adding 500 microliters of 70% EtOH to the column and then centrifuged for 8 minutes. This step was repeated after discarding the flow through. The columns were then centrifuged for a final time for five minutes to remove residual ethanol. The collection tubes were discarded and the columns were placed in 1.5 mL microcentrifuge tubes that were labelled with the ID and date. 100 microliters of pure sterile H2O was added to each tube, which were allowed to stand for 5 minutes and then centrifuged for 2 minutes at 15,000rpm to elute DNA.