Maxiprep

Maxiprep

DO THESE THINGS FIRST: Wear gloves when handling the bacterial culture. Get TENS buffer, 3 M sodium acetate buffer (pH 5.2), vortex, adaptors, 50 mL Falcon tubes, a 500 mL bottle, one 50 mL and one 10 mL graduated cylinder & yellow rotors & a 500mL waste jar (all found in cabinet A) & place the materials on a cart. Label the Falcon tubes accordingly. Manipulation of PCI and chloroform must be done in the fume hood.

***The first step is time sensitive, make sure you are prepared before removing bacterial cultures from the shaker/incubator.***

*Steps 1-4 can be done in the centrifuge room on the second floor.

1. In the large centrifuge room on the second floor (with cart and bacterial cultures), transfer 40 mL of the bacterial culture into a Falcon tube. Spin at 5,000 g for 3 minutes. Discard supernatant into waste jar. Repeat with the rest of the bacterial culture in the same tube. [brought 4 solutions, 12+ falcon tubes, pipette, yellow rotors, and 500 ml waste beaker]

2. Add ~15 mL of TENS buffer. Vortex (on a high setting) for 10 seconds or until the pellet is completely dissolved.

3. Add ~7.5 mL of 3 M sodium acetate pH 5.2. Vortex for 10 seconds.

4. Centrifuge for 5 minutes at 10,000 g and transfer supernatant to a new labeled Falcon tube.

5. Add 10 µL of RNase (Enzyme Rack, Freezer A) and wait for 20 minutes.

6. Add about 5 mL of PCI (Fridge A) from the bottom layer (to avoid getting buffer in pipette). Vortex for 10 seconds and centrifuge at 10,000 g for 5 minutes. [do this in the fume hood]

7. Transfer the top layer to a new, previously labeled Falcon tube (top layer contains DNA) (rather leave some top in the Og tube instead of getting some of the waste/bottom layer). Add about 5 mL of chloroform (Fridge A) to the new tube with the top layer. Vortex for 10 seconds and centrifuge at 10,000 g for 5 minutes. [leave tubes with bottom layer in the fume hood] (0.1 ng/ml had only 15 ml of top layer transferred) (we got bubbles after vortexing with chloroform in the tubes [tube3])

8. Transfer the top layer to a new, labeled Falcon tube. Add an equal volume of 100% ethanol (found in Fridge A) and centrifuge at 10,000 g for 5 minutes. [hope to see a pellet, probably will not. If no pellet, If no pellet, go to step D #1. https://sites.google.com/atsu.edu/norimatsu-lab/protocols/molecular-biology/tens-prep-and-ethanol-precipitation].

9. Carefully decant supernatant into the ethanol waste bottle. Add 40 mL of 70% ethanol (also found in Fridge A) and centrifuge at 10,000 g for 2 minutes.

10. Carefully decant supernatant in the ethanol waste bottle and let DNA dry. Draw a circle on the outside of the tube where the DNA pellet is. Leave the tubes uncapped and place a Kimwipe over the top.

11. Transfer phenol and chloroform wastes from all the tubes into the appropriate waste bottle.

*The Waste jar and bacterial culture flasks from step 1 will need to be autoclaved.

*Falcon tubes that contained TENS buffer and sodium acetate from steps 3-4 can be discarded in the trash.

*Falcon tubes that contained phenol and chloroform need to remain in the fume hood until dry. Once dry they can be discarded in the trash (steps 6-7)

*Pipette tips from phenol and chloroform steps need to be placed in tip waste beaker inside of fume hood. Tips need to be disposed of when dry.

12. Re-suspend the dry DNA in 1 mL of MBG water (Fridge B), vortex, and transfer to a previously labeled 1.5 mL tube. (issues with the pipette leaking made it hard to get all liquid into the 1.5 mL tubes, so there was a drop or two of liquid left in each Falcon tube.)