TENS Prep and Ethanol Precipitation

V. TENS Prep and Ethanol Precipitation: Maxiprep, Midiprep, Miniprep

A. Maxiprep

NOTE: You must wear gloves to handle the bacterial culture. Gather 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. All materials can be 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.

*Please note 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. Head to 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.

2. Add about 15 mL of TENS buffer. Vortex for 10 seconds or until the pellet is dissolved.

3. Add about 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, previously labeled Falcon tube.

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

6. Add about 5 mL of PCl (Fridge A) from the bottom layer. Vortex for 10 seconds and centrifuge at 10,000 g for 5 minutes. *CAUTION: During the vortexing, do not splash into the lid. Let it swirl below the lid.

7. Transfer the top layer to a new, previously labeled Falcon tube. Add about 5 mL of chloroform (Fridge A). Vortex for 10 seconds and centrifuge at 10,000 g for 5 minutes.

8. Transfer the top layer to a new, previously labeled Falcon tube. Add an equal volume of 100% ethanol (Fridge A) and centrifuge at 10,000 g for 5 minutes.

9. Carefully decant supernatant into the ethanol waste bottle. Add 40 mL of 70% ethanol (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 tubes into the appropriate waste bottle. 

*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.

B. Midiprep

NOTE: You must wear gloves to handle the bacterial culture. Gather TENS buffer, 3M sodium acetate buffer (pH 5.2), vortex, adaptors, 14 mL Falcon tubes and a 250 mL bottle. All materials can be 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.

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

1. Transfer cultures into 14 mL Falcon tubes and spin at 5,000 g for 3 minutes. Discard supernatant into waste jar.

2. Add about 2 mL of TENS buffer. Vortex for 10 seconds or until the pellet is dissolved.

Might need to add more

3. Add about 1 mL of 3 M sodium acetate pH 5.2. Vortex for 10 seconds. 

4. Centrifuge for 3 minutes at 5,000 g and transfer supernatant to new, previously labeled Falcon tube. 

5. Add 2.5 µL of RNase (Enzyme Rack, Freezer A) and wait for 5 minutes. 

6. Add about 1 mL of PCl (Fridge A) from the bottom layer. Vortex for 10 seconds and centrifuge at 5,000 g for 5 minutes.

7. Transfer the top layer to a new, previously labeled Falcon tube. Add about 1 mL of chloroform (Fridge A). Vortex for 10 seconds and centrifuge at 5,000 g for 3 minutes.

8. Transfer the top layer to a new, previously labeled Falcon tube. Add an equal volume of 100% ethanol (Fridge A) and centrifuge at 5,000 g for 5 minutes.

9. Carefully decant supernatant into the ethanol waste bottle. Add 5 mL of 70% ethanol (Fridge A) and centrifuge at 5,000 g for 2 minutes. 

10. Carefully decant supernatant into the ethanol waste bottle and let DNA dry. 

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

*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 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 20 µL of MBG water (Fridge A), vortex, and transfer to a previously labeled 1.5 mL tube.

C. Miniprep

NOTE: You must wear gloves to handle the bacterial culture. Gather TENS buffer, 3M sodium acetate buffer (pH 5.2), vortex, 2 mL tubes, and a 250 mL bottle. Label the 2 mL tubes accordingly. All materials can be found in Cabinet A. Manipulation of PCI and chloroform must be done in the fume hood.

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

1. Transfer 2 mL of the bacterial culture into 2 mL tubes and spin at 13,000 rpm for 1 minute. Discard supernatant into waste jar, leaving about 50-100 µL in the tube. Repeat this step if there are 4 mL of bacterial culture.

2. Add 300 µL of TENS buffer. Vortex for 10 seconds or until the pellet is dissolved.

3. Add 150 µL of 3 M sodium acetate buffer pH 5.2. Vortex for 10 seconds. 

4. Centrifuge for 5 minutes at 13,000 rpm and transfer supernatant to new, previously labeled 1.5 mL tube. 

5. Add 1.5 µL of RNAse (Enzyme Rack, Freezer A) and wait for 5 minutes. 

6. Add 450 µL of PCl (Fridge A) from the bottom layer. Vortex for 10 seconds and centrifuge at 13,000 rpm for 5 minutes. Use PCI under the fume hood. Dispose of pipet tip in chemical tip waste bin (located in fume hood).

7. Transfer the top layer to a new, previously labeled 1.5 mL tube. Add 450 µL of chloroform (Fridge A). Vortex for 10 seconds and centrifuge at 13,000 rpm for 5 minutes.

8. Transfer the top layer to a new, previously labeled 1.5 mL tube. Add an equal volume of 100% ethanol (Fridge A) and centrifuge at 13,000 rpm for 5 minutes.

9. Carefully decant the supernatant into the ethanol waste bottle. Add 1 mL of 70% ethanol (Fridge A) and centrifuge at 13,000 rpm for 2 minutes. 

10. Carefully decant the supernatant into the ethanol waste bottle and let DNA dry. Draw a circle on the outside of the tube where the DNA pellet is. If DNA pellet doesn’t form right away, let it stand.

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

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

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

*tubes that contained phenol and chloroform need 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 dried DNA in an appropriate volume of MBG water (Fridge A), vortex, and transfer to a previously labeled 1.5 mL tube.

NOTE: If the DNA solution is too dilute after doing either of the two procedures above, follow the procedure below and re-suspend the DNA in less MBG water.

D. Ethanol Precipitation of Dilute DNA

1. Estimate the volume of the DNA sample. Add 1/10 of the estimated volume in 3 M sodium acetate buffer pH 5.2 (Cabinet A) and mix by inverting 10 times. 

2. Estimate the new volume of the DNA sample. Add 2 times the estimated volume in 100% ethanol (Fridge A), and spin at 13,000 rpm for 5 minutes.

3. Carefully decant supernatant down the drain by turning the tube(s) until completely upside down. Touch the tube to a Kimwipe to wick away any residual ethanol. Add 1 mL 70% ethanol (Fridge A) and centrifuge at 13,000 rpm for 1 minute.

4. Carefully decant supernatant down the drain following the same procedure as step 3. Leave the tubes uncapped and place a Kimwipe over the top.

5. Re-suspend the dry DNA pellet in the appropriate volume of water for a higher concentration.