Lab 8

In this Lab we prepared sampled of M. Gattatus for Gel electrophoresis by doing the following:

1. Collect 3 2.0 mL tubes and label each with respective sample code and add 3 2 mm stainless steel beads to each tube.
2. Take samples of leaves and place one portion into each 2.0mL tube, clean forceps in between each sample transferred.
3. Load tubes into modified saw rack and mount onto the saw. Turn saw on to speed of 3 for 40 seconds.
4. Spin tubes down in centrifuge for 15-20 seconds.
5. Add 434µL of grind buffer to each tube and incubate in water bath at 65˚C for 10 minutes. Make sure to invert each tube every 3 minutes.
6. Add 130µL of 3M (pH 4.7) Potassium Acetate, invert tubes several times and chill on ice for 5 minutes.
7. After the 5 minutes are up, spin tubes on centrifuge for 20 minutes at 15,000 RPM.
8. While tubes are spinning, label three new 1.5mL with appropriate sample ID. After tubes are done spinning transfer supernatant to new tubes, try to avoid drawing up precipitate.
9. Add 1.5 volumes of binding buffer. In this case 600µL were added based off a collected 400µL sample supernatant.
10. Add 650µL from step 9 to Epoch spin column tubes and centrifuge for 10 minutes at 15,000 RPM, discard flow-through in biohazard container. Repeat this again with remaining volume from step 9.
11. Wash the DNA bound silica membrane with 500µL of 70% EtOH and centrifuge again for 8 minutes at 15,000 RPM twice. Discard flow-through each time.
12. Place columns in sterile 1.5mL micro-centrifuge tubes and be sure to label with appropriate sample code.
13. Add 100µL of 65˚ C H2O to each tube, let stand for 5 minutes then centrifuge for 2 minutes at 15,000 RPM to elute DNA.