Mutagenesis - Bochner-Maloy Selection of Tetracycline Sensitivity


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 TetS colonies are seen.


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


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.

Souce: NovoPro    2020-10-07