Genetics - Preparation of Bacteriophage Mu from Dilysogens

Jeff Lawrence (G30)

1. Grow culture of dilysogen overnight at 30° in rich media with appropriate antibiotic.

2. Add 100 µL culture to 10 mL of LB + 5mM CaCl2 in a 125 mL shake flask. Grow at 30° with shaking until about Klett 85, or OD600 of 0.3. Less dense cultures lyse better; I find Klett 95 cultures rarely lyse.

3. Transfer flask to 44° water bath (for rapid temperature shift) and incubate with shaking for 20 to 40 minutes, depending on the strain.

4. Transfer flask to 37° shaker and incubate until lysis occurs. The Klett reading will increase until over Klett 200, then decrease until around Klett 40 or below.

5. Add 100 µL chloroform to flask and incubate at 37° for 5 min. Chill flask on ice.

6. Spin out debris at 8 Krpm for 10 min. Transfer supernatant to a sterile tube.

7. Store at 4° for 1-2 weeks. Titer drops steadily and lysate is nearly unusable after a month. Do not add additional chloroform.
Last Update: Thursday, 19-Jun-2014 11:54:45 PDT
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