Griffitts:Triparental Mating: Difference between revisions
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==Getting the strains growing== | ==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: | * On the day prior to setting up the mating, patch out the three strains involved (from single colonies) onto selective LB plates: | ||
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* With a wooden applicator stick, scrape each strain and separately resuspend as dense suspensions in 200 μL liquid LB | * 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: | * 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 [[Griffitts:Common_media#LB Broth (1 L)|LB]] plate and spread | * Transfer 100 μL of this mixture onto a plain [[Griffitts:Common_media#LB Broth (1 L)|LB]] plate and spread | ||
* Incubate at 30°C for 12 | * Incubate at 30°C for 12–24 hours | ||
===Recovery=== | ===Recovery=== | ||
* To the lawn of cells add 3 mL of liquid [[Griffitts:Common_media#LB Broth (1 L)|LB]]-10% glycerol | * To the lawn of cells add 3 mL of liquid [[Griffitts:Common_media#LB Broth (1 L)|LB]]-10% glycerol | ||
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* Dilute the concentrated mating mix (above) 10X, 100X, and 1000X into [[Griffitts:Common_media#LB Broth (1 L)|LB]] broth | * Dilute the concentrated mating mix (above) 10X, 100X, and 1000X into [[Griffitts:Common_media#LB Broth (1 L)|LB]] broth | ||
* Plate 100 μL of each onto selective [[Griffitts:Common_media#LB Broth (1 L)|LB]] plates (e.g. [[Griffitts:Common_media#LB Broth (1 L)|LB]]-[[Griffitts:Stock solutions#Streptomycin (40 mL)|Sm]]-[[Griffitts:Stock solutions#Neomycin (40 mL)|Nm]]) | * Plate 100 μL of each onto selective [[Griffitts:Common_media#LB Broth (1 L)|LB]] plates (e.g. [[Griffitts:Common_media#LB Broth (1 L)|LB]]-[[Griffitts:Stock solutions#Streptomycin (40 mL)|Sm]]-[[Griffitts:Stock solutions#Neomycin (40 mL)|Nm]]) | ||
* Incubate at 30°C for 24 | * Incubate at 30°C for 24–48 hours<br> | ||
Note: After a couple of days, you should see transconjugants growing, often with an accompanying background haze of cells. Transconjugants must be restreaked to singles to get rid of these would-be hijackers. | 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== | ==Agar-based method== | ||
===Mating "X"=== | ===Mating "X"=== | ||
* Using toothpicks, swipe each of the two ''E. coli'' strains onto a plain [[Griffitts: | * Using toothpicks, swipe each of the two ''E. coli'' strains onto a plain [[Griffitts:Common media#LB Broth (1 L)|LB]] plate, forming an “X” | ||
* On the middle of the “X” smear a large amount of the recipient ''Sinorhizobium'' strain | * On the middle of the “X” smear a large amount of the recipient ''Sinorhizobium'' strain | ||
* Incubate at 30°C for 24 hours | * Incubate at 30°C for 24 hours | ||
[[Image:Triparental Mating Diagram.png|thumb|none|800px|The steps of a triparental mating.]] | |||
===Recovery and Selection=== | ===Recovery and Selection=== | ||
* Using a toothpick pick up a glob of bacteria from the zone where the mating occurred | * Using a toothpick pick up a glob of bacteria from the zone where the mating occurred | ||
* Streak onto the appropriate selective medium | * Streak onto the appropriate selective medium | ||
* Incubate at 30°C for 24 | * Incubate at 30°C for 24–48 hours | ||
* Recover | * Recover single colonies onto the appropriate selective medium | ||
Revision as of 23:54, 23 September 2009
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:
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
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
