From 2007.igem.org

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Filter Conjugation Protocol

We are using conjugation as a means of transferring a plasmid from transformed E.coli into S.oneidensis. The plasmid contains kanamycin resistance, and the S.oneidensis have gentamicin resistance. Thus, a double selection is used to select for S.oneidensis with plasmid (resistant to both kanamycin and gentamicin). During this double selection, the gentamicin will kill any E.coli and the kanamycin will kill S.oneidensis that lacks plasmid.

Add 0.75mL of magnesium sulfate (MgSO4) to a centrifuge tube filter.

Add 9.3 microliters of the donor cells (E.coli BW20767) and 9.3 microliters of the acceptor cells (S.oneidensis AS-92) to the centrifuge tube filter.

Centrifuge until all of the MgSO4 elutes from the column through the filter.

The filter is now covered with the condensed pellet of cells (thus encouraging conjugation)

Remove the filter from the centrifuge tube filter with sterile tweezers.

Incubate the filter (cell-side up) on an LB agar plate for at least 5 hours at 30 degrees C.

Add 1 mL of LB broth to a tube.

Add the filter (now with grown out cell colonies) into the LB broth.

Vortex the tube to release the cell colonies from the filter surface.

Plate the liquid cell stock onto a double selection(kanamycin and gentamicin) plate.

(DS)