Mutagenesis - Bochner-Maloy Selection of Tetracycline Sensitivity
Modification of Jeff Lawrence (X10)
1. Prepare media in two flasks:
12 g Agar
4 g Tryptone
4 g Yeast Extract
40 mg Anhydrotetracycline
400 mL Water
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.
7. Incubate at 30° or higher for 18 - 24 hours until TetS colonies are seen.
- 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.
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.
Last Update: Thursday, 19-Jun-2014 11:54:47 PDT
This page has been viewed 294 times.
eckofoid at ucdavis.edu