November 17, 2021 STARTED AT 3:20 PM

Materials:

TENS buffer

3M sodium acetate buffer (pH 5.2)

Vortex

Adaptors

50 mL Falcon tubes (x4) LABEL THEM

500 mL bottle

50 mL graduated cylinder

10 mL graduated cylinder

1. Go to centrifuge room on the second floor with the cart and bacterial cultures

2. Transfer 40 mL of each bacterial culture into a Falcon tube

3. Spin at 5000g for 3 minutes

4. Discard supernatant into waste jar

5. Repeat with the rest of the bacterial culture in the same tube

6. Add ~15 mL of TENS buffer and vortex for 10 seconds or until pellet is dissolved

7. Add ~7.5 mL of 3M sodium acetate pH 5.2 and vortex for 10 seconds

8. Centrifuge for 5 minutes at 10,000g (label new Falcon tube) and transfer supernatant to new Falcon tube

9. Add 10 µL of RNase (Freezer A on enzyme rack) and wait for 20 minutes

10. Add ~5 mL of PCI (Fridge A) from the BOTTOM LAYER

    1. During the addition of PCI, ~5 mL of the positive control solution was spilled.

11. Vortex for 10 seconds, then centrifuge at 10,000g for 5 minutes

• Label new Falcon tube while waiting

12. Transfer the top layer (no bottom layer!) to the new Falcon tube

13. Add 5 mL of chloroform (Fridge A)

14. Vortex for 10 seconds and centrifuge at 10,000g for 5 minutes

• Label new Falcon tube while waiting

15. Transfer top layer to the new Falcon tube

16. Add an equal volume of 100% ethanol (Fridge A) and centrifuge at 10,000g for 2 minutes

    1. A pellet did precipitate, but we chose to precipitate more DNA from the solution.

17. Pipette 1/10 of the original volume as sodium acetate into the tube

18. Leave to sit on window sill for more extraction

19. Take the phenol/PCI and chloroform wastes from all tubes into the appropriate waste bottles

• TENS buffer stuff and sodium acetate stuff can be thrown in trash

• Falcon tubes that had phenol and chloroform need to remain in the fume hood until they are dry; once dry, can be thrown in trash

• Pipette tips from phenol and chloroform need to be put in tip waste beaker inside of fume hood, then disposed of when dry

Initial Pellets