Griffitts:Triparental Mating

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Contents

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)

Broth-based method

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

  • 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

Note: The mating mixes can be frozen away at this point

Selection

  • 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

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.

Agar-based method

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
The steps of a triparental mating.
The steps of a triparental mating.

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
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