Mutagenesis by Diethylsulfate (DES)
The mutagen DES acts as an alkylating agent which reacts chemically with DNA to modify base (primarily guanine). The resulting damage can lead to errors in DNA replication and repair. This in turn greatly increases the frequency of mutant cells in a bacterial population.
1. Add 2 drops of DES ta a 10 ml screw-capped tube containing 5 ml of E medium with no carbon source.
2. DON'T MOUTH PIPETTE DES.
3. Tighten top and vortex tube; let sit for 10 minutes in 37oC waterbath for DES to dissolve and droplet of excess DES to settle out. ( The goal here is to make a saturated solution of DES).
4. Add 0.1 ml of a full bacterial culture (109 cells/ml) to the aqueous phase and incubate 30-45 minutes at 37o. (Don't shake tube up when adding cells).
5. Remove 0.1 ml aliquots from the DES tube and innoculate 5 ml cultures of nutrient broth . Do a bunch. You want to keep only one mutant of a specific type from each over night culture to avoid sibs.
6. Grow up overnight. This growth allow treated cells to recover and mutant chromosomes to segregate from other (nonmutant) chromosomes which may have been present in cell at time mutations occurred. After overnight growth, culture will contain ~109 cells/ml.
7. Next day dilute culture 106 fold (see serial dilution procedure) and plate 0.1 ml on each of 10 nutrient agar plates using a glass spreader. Incubate plates at 37oC until colonies (~100) appear. These arrays of colonies will be replica printed to nutrient broth and to minimal plates to auxotrophic mutants.
Freq of hitting a gene is 1/1000
Last Update: Thursday, 19-Jun-2014 12:00:23 PDT
This page has been viewed 336 times.
eckofoid at ucdavis.edu