Genetics - B12 Bioassays
Jeff Lawrence (G51)
1. Grow overnight culture of LD336 indicator strain in 2 mL LB.
2. Grow 2 mL (aerobic) or 4 mL (anaerobic) overnight cultures of tester strains in the appropriate minimal medium.
3. Microfuge 1.5 mL of LD336 culture for 3 min. Aspirate supernatant. Resuspend pellet in 1 mL of B12 Buffer:
10 mM Tris, pH 8.0 1 mL 1 M Tris, pH 8.0
10 mM NaCl 200 mL 5 M NaCl
99 mL Sterile water
4. Microfuge for 3 min. Resuspend pellet in 1 mL of B12 Buffer. Save on ice as indicator cells.
5. Microfuge 1.5 mL of tester strains. For anaerobic cultures, refill the tubes as necessary to accumulate sufficient numbers of cells.
6. Aspirate supernatant. Resuspend pellet in 1 mL of B12 Buffer.
7. Microfuge for 3 min. Resuspend pellet in an equal volume of B12 Buffer, typically 10 - 15 µL.
8. Boil tester strains for 10 min.
9. Microfuge for 3 min in the cold room. This procedure yields the cell extract.
10. Spread 100 µL of indicator cells onto an E-Glu-His plate. LD336 is metE Δ(his cob) and will grow only when B12 or methionine is applied to the plate.
11. Place 4 sterile filter-paper discs on the surface of the plate, equally spaced.
12. Immediately apply 10 mL of the supernatant cell extract to a sterile disc. Apply only one extract per disc.
13. Apply 5 nmol of B12 to a single disc on one plate as a control.
14. Incubate plate overnight at 37°, media-side up (else the disc may fall off).
15. Score the plates the next day. If B12 is in the extract, a growth-ring will appear around the sterile filter disc. The amount of B12 may be quantitated by measuring the width of the growth ring.