Jo Bro

Hi all, I'm writing on here as a last-ditch attempt to find some help with a practical class I run for some university students. This was a lab I took over from, and the academic that designed it is no longer with us, so I can't ask him for help! We run a Biochemistry lab that allows students to investigate constitutive mutants for the Lac operon. We get them to select, isolate, enrich, and characterise mutants by growing ML30 in either phenyl-ß-galactosidase, glucose, or lactose. They then grow them on succinate and X-gal plates to visualise the mutations. Every year this lab has problems, and we need to get to the bottom of them, so I was hoping someone may have experience mutating E.coli ML30. Our PG cultures did not grow very well this year, and the flasks were still clear. However, they still grew on the plates, but we had very few mutants, and many students had none. I can't even find out why we are using succinate agar to grow the bacteria after selection, it doesn't affect the Lac operon, so this is maybe why, but I wonder if there is a better medium to use. I really appreciate any help :)