- _1-3: Grow and pellet bugs_
- 4a: Resuspend bacteria in P1 at 75 mg/ml
- 4b: Take 60 ml of suspension _(three reactions)_
- 4c: Split into two batches of 30 ml *(Subsequent steps are in duplicate)*
- 5a: Add 30 ml P2, mix well
- 5b: Incubate @ R__T x 5 min
- 6a: Add 30 ml chilled P3, mix well
- 6b: Incubate @ 0 C x 10 min
- 7: Spin @ >20 kg (J__A-14 @ 14 krpm) x 30 min @ 4C
- 8a: Filter lysate through a prewetted filter _(may need to change filter?)_
- 8b: Wash filter with 10 ml T__E
- 9a: Add 0.6 volumes (60 ml) isopropanol
- 9b: Spin @ 15 kg (J__A-14 @ 12.5 krpm) x 30 min @ 4C
- 10a: Wash with 7.5 ml 70% Et__O__H
- 10b: Spin @ 15 kg (J__A-14 @ 12.5 krpm) x 30 min @ 4C
- 11a: Dry pellet
- 11b: Dissolve in 9.2 ml E__X
- 11c: Combine the two batches, giving 18.4 ml total *(Subsequent steps are in singlicate)*
- 12a: Add 400 ul exonuclease and 1200 ul A__T__P
- 12b: Incubate @ 37 C x >1 hr
- 13: Equilibrate three Q__Tip-500s with 10 ml Q__B__T each
- 14a: Add 10 ml E__X
- 14b: Add 30 ml Q__S
- 14c: Split into three batches of 20 ml *(subsequent steps are in triplicate)*
- _15-19: As normal_
This protocol includes the ModificationWithFilterWash.
CategoryBiology