Mutagenesis - Bochner-Maloy Selection of Tetracycline Sensitivity

Protocol

Modification of Jeff Lawrence (X10)

1. Prepare media in two flasks:

	Flask A
	12 g	Agar
	4 g	Tryptone
	4 g	Yeast Extract
	40 mg	Anhydrotetracycline 
	400 mL	Water

	Flask B
	8 g	NaCl
	8 g	NaH₂PO₄
	400 mL	Water

2. Autoclave media for 20 min. Cool to pouring temperature.

3. Add 9.6 mg Fusaric Acid in 500 µL dimethylformamide to Flask A.

4. Add 4 mL of 20 mM ZnCl₂ to Flask B.

5. Mix and pour. Use plates within 36 hours.

6. Grow up strains overnight in LB-tet media to fully induce the operon.

6. Quantities:

For full Tn10 : Plate 100 µL of a 10^-2 dilution of a fresh overnight culture.

For Tn10dTet : Plate 200 µL of a fresh overnight culture.

7. Incubate at 30° or higher for 18 - 24 hours until Tet^S colonies are seen.

Comments

The original reciped called for chlortetracycline (Kelly Hughes, personal communication, 2013) in place of anhydrotetracycline, in which case incubation had to be at 42°.

References

Bochner, B. R., Huang, H. C., Schieven, G. L., & Ames, B. N. (1980). Positive selection for loss of tetracycline resistance. Journal of bacteriology, 143(2), 926-933.

Maloy, S. R., & Nunn, W. D. (1981). Selection for loss of tetracycline resistance by Escherichia coli. Journal of bacteriology, 145(2), 1110-1111.

Kelly Hughes, personal communication, 2013.

Last Update: Thursday, 12-Dec-2024 00:55:27 UTC

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Eric Kofoid eckofoid at ucdavis.edu