7. Place membrane filter on an EMJH media plate with 0.3 mM
DAP (without kanamycin) with the bacteria-containing side
facing up/away from the media (Fig. 3) (see Notes 25 and 26).
8. Repeat this filtration procedure with additional media and
membrane filters until Leptospira-containing EMJH media is
exhausted (see Note 27).
Fig. 2 Removing membrane from glass frit filter with stopper. Note how 15 mL
funnel is placed at 45
angle to frit for purchase while removing membrane filter
so that membrane filter doesn’t jump due to static or vacuum pressure. (Photo by
Natalia Lizardo, Manhattanville College)
Fig. 3 How to position seven membrane filters on one EMJH solid media plate.
Bacteria deposited on membrane filter are visible as a circular sheen in the
middle of each membrane filter. No bubbles are present between membrane
filters and solid media to allow transfer of constituents to bacteria during
conjugation
Random Transposon Mutant Library
83
DAP (without kanamycin) with the bacteria-containing side
facing up/away from the media (Fig. 3) (see Notes 25 and 26).
8. Repeat this filtration procedure with additional media and
membrane filters until Leptospira-containing EMJH media is
exhausted (see Note 27).
Fig. 2 Removing membrane from glass frit filter with stopper. Note how 15 mL
funnel is placed at 45
angle to frit for purchase while removing membrane filter
so that membrane filter doesn’t jump due to static or vacuum pressure. (Photo by
Natalia Lizardo, Manhattanville College)
Fig. 3 How to position seven membrane filters on one EMJH solid media plate.
Bacteria deposited on membrane filter are visible as a circular sheen in the
middle of each membrane filter. No bubbles are present between membrane
filters and solid media to allow transfer of constituents to bacteria during
conjugation
Random Transposon Mutant Library
83