Mutagenesis - Bacterial Cells with DES


Jeff Lawrence (X01)

Pilot experiment - Perform a pilot for any new strain of bacteria to be mutagenized

1. Grow bacterial culture overnight

2. Add 2 drops of Diethyl Sulfate to 5 mL E Medium in a 10 mL screw-capped tube.

3. Vortex tube. Incubate at 37° for 10 min.

4. Layer 100 µL of an overnight bacterial culture to the top of the tube. DO NOT mix.

5. Immediately remove 100 µL from the top of the tube and place in 5 mL LB.

6. Remove additional 100 µL aliquots at 1, 2, 4, 8, 16, 32, and 48 min. and place each in a separate tube of 5 mL of LB. Grow cultures overnight.

7. Dilute each culture 106-fold to 2x103 cells/mL. Plate 50, 100, and 200 mL aliquots of each dilution on LB plates. Grow plates overnight.

6. Replica print plates with about 200 colonies to rich medium and minimal medium.

7. Successful mutagenesis will yield approximately 1% auxotrophic colonies, corresponding to 5 mutations per genome. Under these condition, 1/1000 cells will be mutant in any one gene. Choose the time allowing for this number of auxotrophs

Full-scale experiment - Perform this protocol for isolation of independent mutations

1. Grow bacterial culture overnight

2. Add 2 drops of Diethyl Sulfate to 5 mL E Medium in a 10 mL screw-capped tube.

3. Vortex tube. Incubate at 37° for 10 min.

3. Layer 100 µL of an overnight bacterial culture to the top of the tube. DO NOT mix. Incubate at 37° for the length of time determined above (typically between 5 and 45 minutes).

4. Transfer 100 µL aliquots to 5 mL LB. Inoculate 1 tube for each independent mutant.

5. Grow culture overnight. Dilute culture 106-fold to 2x103 cells/mL. Plate 100 µL aliquots on 10 LB plates for each independent mutant desired.

6. Grow plates overnight. Replica print to media to screen for auxotrophs and desired mutant phenotypes.

7. Freeze cultures of independent replicates.
Last Update: Thursday, 19-Jun-2014 11:54:46 PDT
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Eric Kofoid eckofoid at ucdavis.edu