Erickson:Tri-parental Mating
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(New page: Tri-parental Mating 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) 2. Combine 200 µL of...) |
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Tri-parental Mating | 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) | 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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2. Combine 200 µL of the donor, helper, and recipient strains into one centrifuge tube | 2. Combine 200 µL of the donor, helper, and recipient strains into one centrifuge tube | ||
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3. Centrifuge and remove supernatant | 3. Centrifuge and remove supernatant | ||
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4. Re-suspend pellet in 25 µL broth | 4. Re-suspend pellet in 25 µL broth | ||
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5. Spot onto BHI plate (no antibiotics) | 5. Spot onto BHI plate (no antibiotics) | ||
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6. Incubate overnight at 30°C | 6. Incubate overnight at 30°C | ||
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7. Scrape a colony from plate and re-suspend in broth | 7. Scrape a colony from plate and re-suspend in broth | ||
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8. Dilute each solution to 10^-1 | 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 | 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 | 10. Incubate overnight at 30°C | ||
Revision as of 14:36, 23 June 2008
Tri-parental Mating
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)
2. Combine 200 µL of the donor, helper, and recipient strains into one centrifuge tube
3. Centrifuge and remove supernatant
4. Re-suspend pellet in 25 µL broth
5. Spot onto BHI plate (no antibiotics)
6. Incubate overnight at 30°C
7. Scrape a colony from plate and re-suspend in broth
8. Dilute each solution to 10^-1
9. Plate 100 uL of each solution onto a BHI plate with kanamycin and irgasan
10. Incubate overnight at 30°C
