TITLE: Wisecaver Lab CTAB-based extraction of high molecular weight DNA from photosynthetic sea slugs 
AUTHORS: Jennifer H Wisecaver, Raeya Ogas, Pax Tomko, Gregory Gavelis

[DESCRIPTION]
Protocol for CTAB-based extraction of high molecular weight DNA from  photosynthetic sea slugs. This protocol is optimized for samples with high mucopolysaccharide contents and is derived from:

Extraction of high molecular weight DNA from molluscs. 1993. Trends in Genetics.Vol 9: pg 407
https://doi.org/10.1016/0168-9525(93)90102-N.

[STEPS]
SECTION: Prepare CTAB DNA extraction buffer
1. Prepare 100 mL CTAB buffer:
100 mL Ultrapure distilled water
1.576 g TrisHCL ph 8 (100 mM)
0.584 g EDTA (20 mM)
8.82 g NaCl (1.4 M)
10 mg Proteinase K (0.1 mg/mL)

Note: This stock buffer is sufficient for 9-10 extractions. 
SECTION: Prepare equipment and reagents
4. On the day of the of extraction:
Chill centrifuge to 4 °C
Get liquid nitrogen
Chill mortar and pestle in the -20 °C freezer
Bring Ampure beads to room temperature (~ 20 °C)
Prepare 5 mL of 70% Ethanol
SECTION: Prepare slug sample(s)
5. Decide if performing DNA extraction using whole slugs or dissected tissue. 
SECTION: Prepare CTAB DNA extraction buffer
2. On the day of an extraction, per sample: add 20 µL beta-mercaptoethanol (BME) to 10 mL CTAB buffer. 
 

SECTION: Prepare CTAB DNA extraction buffer
3. Use a water bath to preheat CTAB+BME buffer at 60 °C in a water bath.

SECTION: Prepare slug sample(s)
6. Use mortar + pestle + liquid nitrogen to grind the sample(s), either whole/partial slug or dissected brain tissue) to a powder
SECTION: Remove mucopolysaccharides
7. Transfer pulverized sample to a 50 mL centrifuge tube with 10 mL preheated CTAB+BME buffer

SECTION: Remove mucopolysaccharides
8. Incubate at 60 °C for 30 min

SECTION: Remove mucopolysaccharides
9. Add 10 mL of chloroform:isoamyalcohol. Mix well.

 

SECTION: Remove mucopolysaccharides
10. Centrifuge at 7700 x g for 10 min

SECTION: Remove mucopolysaccharides
11. Transfer 2.5 mL aqueous phase to a 5 mL lo-bind microcentrifuge tube. 

SECTION: Precipitate nucleic acids
12. Add 2/3 volume (1.7 mL) isopropanol and gently invert tube for 5 min at room temperature.

SECTION: Precipitate nucleic acids
13. Centrifuge for   at 10000 x g. Discard th supernatant. 

SECTION: Precipitate nucleic acids
14. Add 300 µL of 70% ethanol to the DNA pellet. Use a pipette tip to dislodge the pellet from the wall of the microcentrifuge tube. 

SECTION: Precipitate nucleic acids
15. Centrifuge for 5 min at 10000 x g. Discard the supernatant.

SECTION: Precipitate nucleic acids
16. Use a pipette to remove the remainder of teh supernatant from the tube. Be careful not to disrupt the pellet. 
SECTION: Precipitate nucleic acids
17. Gently lay the tube on its side on a sterile work surface with the cap open. Allow the tube to air dry in this position for 10 min. 

SECTION: Degrade RNA and extract DNA
18. Resuspend the pellet in 120 µLof TE buffer. 

SECTION: Degrade RNA and extract DNA
19. Add DNAase-free RNase A to a final concentration of 1 ug/mL. Incubate at 37 °C for 30 min. 

SECTION: Degrade RNA and extract DNA
20. Add 120 µL phenol:chloroform:isoamylalcohol.
 


SECTION: Degrade RNA and extract DNA
21. Centrifuge for 10 min at 10000 x g. Transfer 60 µL of the aqueous phase to a clean 1.5 mL lo-bind microcentrifuge tube. Discard the remainder. 

 


SECTION: Bead-purify DNA
22. Resuspend the ampure beads by quickly vortexing. Add 108 µL ampure bead solution to the aqueous phase sample. Mix gently by pipetting up and down 10 times using a wide-bore pipette tip. 

SECTION: Bead-purify DNA
23. Incubate at room temperature (20 °C) for 5 min. 

SECTION: Bead-purify DNA
24. Place tube in the magnetic stand until the solution becomes clear (approx. 2 min).

SECTION: Bead-purify DNA
25. With the tube still in the magnetic stand, carefully discard the clear supernatant
SECTION: Bead-purify DNA
26. Keeping the tube in the magnetic stand, add 200 µL of 70% ethanol. Incubate for 30 s. Gently remove the clear supernatant, being careful not to disturb the beads. 

Repeat this wash step three times. 
SECTION: Bead-purify DNA
27. Ensure that the ethanol is completely removed and leave the tube top open to dry for 5 min. Do not 'overdry' (i.e., to the point that the bead streak is cracking)

SECTION: Bead-purify DNA
28. Remove the tube from the magnetic stand. Add 60 µL TE to dissolve the DNA. 

SECTION: Bead-purify DNA
29. Mix gently by pipetting up and down 10 times using a wide-bore pipette tip. 
SECTION: Bead-purify DNA
30. Incubate at room temperature (20 °C) for 5 min

SECTION: Bead-purify DNA
31. Place tube in the magnetic stand until the solution becomes clear (approx. 1 min).

SECTION: Bead-purify DNA
32. Transfer the clear DNA suspension to a new tube. Discard the old tube with the beads. 
SECTION: QC and cleanup
33. Make sure all chloroform waste is properly disposed of in the chemical fume hood. 
Wipe down the chemical fume hood and the benchtop with 70-75% ethanol. 
SECTION: QC and cleanup
34. Assess DNA integrity using the TapeStation. Make sure you reserve time on the TapeStation Teams calendar. 
SECTION: QC and cleanup
35. While the TapeStation is running, measure the DNA concentration using the Qubit.
SECTION: QC and cleanup
36. If the TapeStation reports a high DNA integrity (DIN) score (≥ 7) and DNA concentration ≥ 20 ng/uL, then you have successfully isolated DNA for whole genome sequencing!
   
Cap your sample tube tightly. Make sure each tube is labeled on the cap and vertically on the side with the sample's unique identifier*. Wrap the cap with parafilm and place sample in the 'Novogene' box in the full sized -20 freezer.   

*The identifier should start with a letter and be no more than 12 characters long. Write it carefully using a fine tip permanent marker as this tube will be shipped off for DNA sequencing.