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.
Souce: NovoPro 2020-10-07