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

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