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 TetS 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, 19-Jun-2014 11:54:47 PDT
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Eric Kofoid eckofoid at ucdavis.edu