Griffitts:Triparental Mating

Getting the strains growing

 * On the day prior to setting up the mating, patch out the three strains involved (from single colonies) onto selective LB plates:
 * A recipient S. meliloti strain (this should be Sm-resistant)
 * The E. coli helper strain (usually MT616 or B001, on LB-Cm)
 * The E. coli donor (usually a plasmid-bearing DH5α strain)

Mating Mix

 * With a wooden applicator stick, scrape each strain and separately resuspend as dense suspensions in 200 μL liquid LB
 * Combine the three strains as follows:
 * 100 μL of the S. meliloti recipient strain
 * 5 μL of the helper strain
 * 5 μL of the donor strain
 * Transfer 100 μL of this mixture onto a plain LB plate and spread
 * Incubate at 30°C for 12–24 hours

Recovery
Note: The mating mixes can be frozen away at this point
 * To the lawn of cells add 3 mL of liquid LB-10% glycerol
 * Use a sterile spreader to resuspend the cells into the LB-10% glycerol
 * Distribute this “mating mix” into microcentrifuge tubes
 * Plate out on selective medium

Selection
Note: After a couple of days, you should see transconjugants growing, often with an accompanying background haze of cells (satellite colonies). Transconjugants must be restreaked to singles to get rid of these would-be hijackers.
 * Dilute the concentrated mating mix (above) 10X, 100X, and 1000X into LB broth
 * Plate 100 μL of each onto selective LB plates (e.g. LB-Sm-Nm)
 * Incubate at 30°C for 24–48 hours

Mating "X"

 * Using toothpicks, swipe each of the two E. coli strains onto a plain LB plate, forming an “X”
 * On the middle of the “X” smear a large amount of the recipient Sinorhizobium strain
 * Incubate at 30°C for 24 hours

Recovery and Selection

 * Using a toothpick pick up a glob of bacteria from the zone where the mating occurred
 * Streak onto the appropriate selective medium
 * Incubate at 30°C for 24–48 hours
 * Recover single colonies onto the appropriate selective medium