Generating Frozen Stocks of Strains

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1. Obtain an overnight culture of M. xanthus during log growth phase. Label a cryovial (2mL) on the top with your strain name, and on the side with strain name, date, your initials, and any information such as resistance markers and/or fluorescence.

2. Spin down 1.5ml of culture in a microfuge tube at max speed for 1 min and pipet off supernatant.

3. To the same tube, add another 1.5mL of culture, spin at max speed for 1 min, and pipet off supernatant.

4. Resuspend cell pellet in 700uL of fresh CTTYE broth very thoroughly by pipetting up and down.

5. Add another 700uL of fresh CTTYE and mix thoroughly but carefully.

6. Transfer the 1.4mL of culture to the labeled cryovial, add 600uL of 40-80% glycerol, and mix gently by pipetting.

7. Drop into liquid nitrogen (do not get liquid nitrogen on your own! ask ahead of time if you are planning on needing it.)

8. After some time in the liquid nitrogen, transfer tubes to the -80C freezer quickly without exposing them to room temperature for more than a few seconds.