a-Complementation Testing of Recombinant Bacteria

1.) To a premade LB agar plate containing the appropriate antibiotics, add 40mL of 20mg/mL X-gal* and 4mL of 200mg/mL IPTG*

2.) Using a sterile glass spreader, spread the solution over the entire surface of the plate

3.) Incubate the plate at 37°C until all the fluid has disappeared (can take up to 3-4 hours)

4.) Inoculate the plate with bacteria, either a colony or a suspension

5.) Incubate the plate in an inverted position for 12-16 hours at 37°C

6.) Store the plate at 4°C for several hours to allow the blue color to develop fully

Blue colonies represent non-recombinant bacteria

White colonies represent recombinants with inactivated β-galactosidase, although they sometimes have a faint blue spot in the center

*X-gal:

20mg X-Gal

1mL dimethylformamide total

*IPTG:

2g IPTG

8mL H₂O total

Filter sterilize