Mutagenesis - Bochner Selection of Tetracycline Sensitivity

Jeff Lawrence (X10)

1. Prepare media in two flasks:

	Flask A
	12 g	Agar
	4 g	Tryptone
	4 g	Yeast Extract
	40 mg	Chlortetracycline
	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 42° for 18 - 24 hours until Tet^S colonies are seen.