35 Bacterial Lysis and Genomic DNA Isolation
Growth of Isolates
3. A colony of each of your isolates will be used to start a liquid culture in Tryptic Soy Broth (TSB).
4. Grow liquid cultures with shaking at 30°C for 16-18 hours.
5. Concentrate 1 ml of culture to 250 µl by resuspending the cell pellet in PBS.
6. Pre-lyse with 1 µl of metapolyzyme for 30 min at 35°C
Bacterial Lysis
33. Spin the PowerBead Pro Tube briefly to ensure that the beads have settled at the bottom. Add up to 250 µl of resuspended bacteria and 800 µl of Solution CD1. Vortex briefly to mix.
34. Secure the PowerBead Pro Tube horizontally on a Vortex Adapter for 1.5–2 ml tubes. Vortex at maximum speed for 10 min. Note: If using the Vortex Adapter for more than 12 preps simultaneously, increase the vortexing time by 5–10 min.
35. Centrifuge the PowerBead Pro Tube at 15,000 x g for 1 min.
36. Transfer the supernatant to a clean 2 ml Microcentrifuge Tube (provided). Note: Expect 500–600 µl. The supernatant may still contain some soil particles.
37. Add 200 µl of Solution CD2 and vortex for 5 s.
38. Centrifuge at 15,000 x g for 1 min at room temperature. Avoiding the pellet, transfer up to 700 µl of supernatant to a clean 2 ml Microcentrifuge Tube (provided). Note: Expect 500–600 µl.
gDNA Binding and Elution
39. Add 600 µl of Solution CD3 and vortex for 5 s.
40. Load 650 µl of the lysate onto an MB Spin Column and centrifuge at 15,000 x g for 1 min.
41. Discard the flow-through and repeat step 8 to ensure that all of the lysate has passed through the MB Spin Column.
42. Carefully place the MB Spin Column into a clean 2 ml Collection Tube (provided). Avoid splashing any flow-through onto the MB Spin Column.
43. Add 500 µl of Solution EA to the MB Spin Column. Centrifuge at 15,000 x g for 1 min.
44. Discard the flow-through and place the MB Spin Column back into the same 2 ml Collection Tube.
45. Add 500 µl of Solution C5 to the MB Spin Column. Centrifuge at 15,000 x g for 1 min.
46. Discard the flow-through and place the MB Spin Column into a new 2 ml Collection Tube (provided).
47. Centrifuge at up to 16,000 x g for 2 min. Carefully place the MB Spin Column into a new 1.5 ml Elution Tube (provided).
48. Add 50–100 µl of Solution C6 to the center of the white filter membrane. 17. Centrifuge at 15,000 x g for 1 min. Discard the MB Spin Column. The DNA is now ready for downstream applications.
