M. xanthus genomic DNA extraction with Zymo (yellow) kit

1. Turn a heat block to 55C.

2. If using an overnight culture, spin down 1.5mL of cells in a microfuge tube. If using cells from a plate, scrape up some culture with a p1000 pipet tip and resuspend it in 500uL of TPM, PBS, or DNA elution buffer from the kit. Spin these cells down at max speed for 1 min to create a pellet.

3. Pour and pipet of supernatant, and resuspend pellet THOROUGHLY in 200uL of TPM, PBS, or DNA elution buffer.

4. Place tube in the freezer for 5-10 minutes until the sample is entirely frozen, then remove from the freezer to thaw.

4. Once thawed, immediately add 200uL of RED Biofluid and Cell buffer, and and 20uL of proteinase K (located in the -20C).

5. Mix thoroughly or vortex for 10-15 seconds, and then incubate 55C for 15 minutes.

6. Add 1 volume of Genomic Binding Buffer to the sample. Mix thoroughly with the pipet for 10-15 seconds. (Ex. if you have 420uL in your sample, add 420uL of Genomic Binding Buffer).

7. Transfer the sample to a spin column in a collection tube. Centrifuge at >12,000xg for 1 min. Discard the collection tube with the flowthrough and place the column into a fresh collection tube.

8. Add 400uL of DNA Pre-Wash buffer to the spin column. Centrifuge at >12,000xg for 1 minute. Empty the collection tube into the small biohazard bin and place the spin column back into the collection tube.

9. Add 700uL of gDNA Wash Buffer to the spin column. Centrifuge at >12,000xg for 1 minute. Empty the collection tube and place it back under the spin column.

10. Add 200uL of gDNA Wash Buffer to the spin column. Centrifuge at >12,000xg for 1 minute. Empty the collection tube and place it back under the spin column.

11. Without adding anything to the spin column, centrifuge at >12,000xg for 1 minute to remove any residual buffer, and discard the collection tube.

12. Place the spin column into a clean, labeled 1.5mL microfuge tube. Label both the top and the side of the microfuge tube. Pipet 50uL of nuclease-free water over the center of the spin column matrix, dropping the water onto the matrix without getting it onto the side of the tube or touching the matrix with the pipet.

13. Incubate at room temperature for 5 minutes and then centrifuge at max speed for 1 minute to elute the DNA from the column. You will not be able to cap the microfuge tube with the spin column in it, but orient the tubes so that the open cap is facing inward toward the center of the rotor.

14. Check DNA concentration on the nanodrop, and then put labeled tube of DNA in the -20C freezer to store until later use.