Lab 8

For Lab 8, I was preparing DNA extractions from Mimulus guttatus leaf samples. I had three samples: CATB JK 01, simplified to JK01, SHOR 002 AJC, simplified to JK02, and TROJ 003 AJC, simplified to JK03. Although broadly similar to the prior fish DNA extraction, plant tissue is more dense, so we first had to pulverize it using steel ball bearings for 40 seconds using a modified reciprocating saw to masticate the tissue. Following mastication, the tubes were spun down to concentrate the tissue powder, and had 434 microliters of warm grind buffer added to each tube. The grindate was then incubated for 10 min in a water bath, after which 130 mL of potassium acetate was added. The tubes were then incubated on ice for 5 minutes to precipitate out DNA. Next, the sample was vortexed at 15000 RPM in a microcentrifuge for 20 minutes to separate the supernatant from the solid leaf tissue particles. Next, the supernatant was transferred to new 1.5 mL sterile tubes labelled with the sample IDs ( JK01, JK02, and JK03). After that,  I added 600 microliters of binding buffer to bind the DNA to a flow-through collection column. I then passed the buffer solution containing the DNA through an Epoch spin column for 2 cycles of concentration in the column followed by two washes with 500 mL 70% EtOH, with a final 5 min spin cycle to remove the remaining EtOH. After this, the columns were moved to the new, labeled 1.5 mL microcentrifuge tubes, and had 100 microliters of sterile H20 added to collect the eluted DNA. After this, they were spun for a final centrifuge cycle for 2 minutes for elution, then taken away by Dr. Paul and stored on ice for later processing.

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