revised 9 July 2003
|
|
|
Now that you understand the practical side of generating large numbers of mutated cells, we will search for two specific types of phenotypes -- cells unable to grow using lactose and those that make ß-galactose in the absence of lactose. Even after mutagenesis, the number of cells that carry the specific types of mutations we are interested is still quite small. It would make our task easier if we could enrich for cells that carrying the types of mutations we are interested in. |
|
To do this, we will perform a selection. After the mutagenesis, we establish overnight cultures in glucose-containing media. The next morning (bright and early), we take a sample from each of our overnight cultures. Dilute the sample 1:20 into fresh media containing lactose as the sole energy/carbon source. Allow those cultures to grow for 60 minutes, then add 0.05 µg/ml ampicillin -- a derivative of the antibiotic penicillin. In the presence of ampicillin, cells that are actively growing will die. Cells unable to grow on lactose will survive preferentially. |
|
|
After 4 hours, plate out the cultures onto glucose/agar plates and let them grow up overnight. Next morning, prepare replica lactose/agar plates and allow them to grow up overnight. Store the original glucose/agar plates in the cold room, you will need them for your screen.
|
|
|
|
We are now ready
to screen for mutations that fail to grow on lactose. We will call these
lac- mutations. To be sure that these are independent mutations, we will
isolate only a one lac- strain from each original UV-mutagenized culture.
|
|
|
|
|
revised 9 July 2003 |
|| |