Erickson:Tri-parental Mating

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Current revision (18:44, 24 April 2009) (view source)
 
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Tri-parental Mating
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*Tri-parental Mating
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1. Grow the donor, helper, and recipient strains of bacteria separately in 3 mL of LB Broth overnight (30° C for Y. pestis and 37°C for E. coli)
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    Grow the donor, helper, and recipient strains of bacteria separately in 3 mL of LB Broth overnight (30° C for Y. pestis and 37°C for E. coli)
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    Combine 200 µL of the donor, helper, and recipient strains into one centrifuge tube
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2. Combine 200 µL of the donor, helper, and recipient strains into one centrifuge tube
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    Centrifuge and remove supernatant
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    Re-suspend pellet in 25 µL broth
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3. Centrifuge and remove supernatant
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    Spot onto BHI plate (no antibiotics)
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    Incubate overnight at 30°C
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4. Re-suspend pellet in 25 µL broth
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    Scrape a colony from plate and re-suspend in broth
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    Dilute each solution to 10^-1
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5. Spot onto BHI plate (no antibiotics)
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    Plate 100 uL of each solution onto a BHI plate with kanamycin and irgasan
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    Incubate overnight at 30°C
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6. Incubate overnight at 30°C
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7. Scrape a colony from plate and re-suspend in broth
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8. Dilute each solution to 10^-1
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9. Plate 100 uL of each solution onto a BHI plate with kanamycin and irgasan
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10. Incubate overnight at 30°C
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Current revision

  • Tri-parental Mating
    Grow the donor, helper, and recipient strains of bacteria separately in 3 mL of LB Broth overnight (30° C for Y. pestis and 37°C for E. coli)
    Combine 200 µL of the donor, helper, and recipient strains into one centrifuge tube
    Centrifuge and remove supernatant
    Re-suspend pellet in 25 µL broth
    Spot onto BHI plate (no antibiotics)
    Incubate overnight at 30°C
    Scrape a colony from plate and re-suspend in broth
    Dilute each solution to 10^-1
    Plate 100 uL of each solution onto a BHI plate with kanamycin and irgasan
    Incubate overnight at 30°C
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