5'FOA Counterselection on M9 5'FOA Glucose plates

References:

Boeke, J. D., F. LaCroute, and G. R. Fink. 1984. A positive selection for mutants lacking orotidine-5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol. Gen. Genet.197:345-346.
Teca Calcagno Galvão, Víctor de Lorenzo. 2005. Adaptation of the Yeast URA3 Selection System to Gram-Negative Bacteria and Generation of a ΔbetCDE Pseudomonas putida Strain. Applied and Environmental Microbiology. Feb 2005, 71 (2) 883-892; DOI: 10.1128/AEM.71.2.883-892.2005

To 170mL dH20 add the following:

50mL 10X NREL M9 salts

1mL MgSO4

50uL CaCl2

25mL stock glucose soln (20% stock so the final is 1%)

250uL trace elements solution from teknova

8mg (+/- 0.5 mg) uracil powder

100mg 5'FOA (Invitrogen or US Biologicals)

Heat gently at ~60C for 1-2 hours to get the FOA to dissolve. A small amount of FOA will not dissolve after 2 hours, which doesn't seem to be an issue? We have also had success with using a 100mg/mL 5'FOA solution from Zymoresearch [catalog number F9003]. Use 1mL of the 5'FOA soln from Zymoresearch if you end up picking that catalog number.

While the FOA is dissolving,

Prepare 250mL 4% molten agar, melt and keep in a 50C waterbath while the 5'FOA mixes. Hint: microwave for 30 minutes with the cap mostly unscrewed at power level 3. Check on it periodically to ensure it doesn't boil over.

Label plates with three red stripes. Combine the M9+glucose+FOA solution with 4% agar and mix well to ensure agar is fully incorporated. Aim for 20mLs per plate. Generates ~1 sleeve (16-20 plates). We've used plates 2-3 months after pouring without issue.