TITLE: Extraction of High Molecular Weight DNA from Aureococcus anophagefferens Virus
AUTHORS: Alex  Truchon, Eric  Gann, Steven W Wilhelm

[DESCRIPTION]
High molecular weight DNA has become a necessary resource with the advancement of long-read sequencing from Nanopore ONT and PacBio SMRT. Often times excessive treatment with phenol-chloroform and prolonged periods of centrifugation results in sheared DNA below 10 kilobases. DNA extracted from viruses is notably less stable than that of cellular organisms, and is often heavily sheared upon extraction. Likewise, kits designed specifically for viral DNA extraction tend to return very low yields of DNA. This protocol seeks to subvert the shearing of typical DNA extractions by combining the process with pulsed-field gel electrophoresis while also maintaining high yields.

[GUIDELINES]
Low melting agarose gels are extremely fragile and take a long time to cast. Casting and moving the gel inside a 4° C walk-in refrigerator can ease the process.

[STEPS]
SECTION: Viral Particle Preparation and Lysis
1. Infect 1 L of Aureococcus anophagefferens CCMP1984 with 10 mL of Aureococcus anophagefferens Virus (AaV) lysate.

SECTION: Viral Particle Preparation and Lysis
2. Incubate cultures at 19 degrees C on a 14:10 light-dark cycle until complete lysis of the culture (~10 days).
SECTION: Viral Particle Preparation and Lysis
3. Concentrate the lysed culture to a final volume of 50 – 100 mL final volume via tangential flow filtration using a 30 kDa filter.
SECTION: Viral Particle Preparation and Lysis
4. Centrifuge concentrated lysate for 10 min at 3,500 xg at 4° C to pellet cellular organisms in lysate.

SECTION: Viral Particle Preparation and Lysis
5. Remove supernatant and discard pellet.
SECTION: Viral Particle Preparation and Lysis
6. Add 10% Triton-X to supernatant to a final concentration of 1%.
SECTION: Viral Particle Preparation and Lysis
7. Centrifuge 50 mL lysate at a speed of 24,000 xG  at 4° C for 75 min to pellet viruses. Discard supernatant.

SECTION: Viral Particle Preparation and Lysis
8. Resuspend the pelleted viruses in a 500 µL of 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2.

SECTION: Viral Particle Preparation and Lysis
9. Add 9 µL DNAse I (2.0 mg/mL) and mix gently.

SECTION: Viral Particle Preparation and Lysis
10. Incubate at room temperature for 60 min.

SECTION: Viral Particle Preparation and Lysis
11. Add 6 µL of 500 mM EDTA (pH 8.0). Mix gently.

SECTION: Viral Particle Preparation and Lysis
12. While incubating, prepare 100 mL 2% molecular grade agarose solution by microwaving agarose/water mixture . 

SECTION: Viral Particle Preparation and Lysis
13. Add 2% agarose solution to concentrated lysate at a 1:1 ratio for a final concentration of 1% agarose. Store in water bath until ready to cast.
SECTION: Viral Particle Preparation and Lysis
14. Cast agarose—lysate mixture in BioRad CHEF Mapper Plug Molds (Catalog No. 1703713) and allow to solidify at room temperature.
SECTION: Viral Particle Preparation and Lysis
15. Prepare viral lysis buffer.
SECTION: Viral Particle Preparation and Lysis
16. After agarose has solidified punch out of molds and add two per Eppendorf tube with 1 mL lysis buffer.

SECTION: Viral Particle Preparation and Lysis
17. Add proteinase-K to a final concentration of 1 mg/ml to each tube.
SECTION: Viral Particle Preparation and Lysis
18. Incubate over night at 37° C under gentle rotation (~ 2-3 xG or 100 rpm) in a shaking incubator.
SECTION: Viral Particle Preparation and Lysis
19. Cast a 2% low-melting agarose gel and allow at least 60 min for gel to solidify.

SECTION: Viral Particle Preparation and Lysis
20. Decant lysis buffer from overnight incubations of plugs.
SECTION: Viral Particle Preparation and Lysis
21. Prepare TNE wash buffer (10 mM Tris-Base, 200 mM NaCL, 0.5 mM EDTA). Filter sterilize through a 0.22 µM syringe filter.
SECTION: Viral Particle Preparation and Lysis
22. Wash with 1 mL TNE buffer by resuspending and shaking at room temperature for 10 min.

SECTION: Viral Particle Preparation and Lysis
23. Pour off TNE buffer.
SECTION: Viral Particle Preparation and Lysis
24. Repeat steps 22-23 once.
SECTION: Viral Particle Preparation and Lysis
25. Carefully insert plugs into individual wells of the low melting agarose gel using a sterile razor blade or pipette tip.
SECTION: Viral Particle Preparation and Lysis
26. Seal the wells by pouring liquid 2% agarose over the wells and allow poured agarose to solidify.
SECTION: Viral Particle Preparation and Lysis
27. Run gel at approximately 120 V for 90 min to120 min or until a high molecular weight band of DNA can be visualized.

SECTION: High Molecular Weight DNA Extraction
28. Excise high molecular weight bands from the agarose under UV light, being careful to remove as little excess agarose as possible. Place excised pieces of gel in Eppendorf tubes.
SECTION: High Molecular Weight DNA Extraction
29. Melt agarose in a water bath set to 80° C Bring volume in tube up to 500 µL with MilliQ water. The melted agarose volume should never exceed 500 µL.

SECTION: High Molecular Weight DNA Extraction
30. Once entirely melted (after at least ten minutes), add1 mL basic phenol to the tube and invert to mix.

SECTION: High Molecular Weight DNA Extraction
31.  Place phenol—agarose mixture at -80° C for 10 min

SECTION: High Molecular Weight DNA Extraction
32. Centrifuge the mixture for 5 min at maximum speed on a microcentrifuge at room temperature.

SECTION: High Molecular Weight DNA Extraction
33. Carefully remove upper aqueous layer and place in a clean 2 mL Eppendorf tube. Discard the lower phenol layer.

SECTION: High Molecular Weight DNA Extraction
34. Add 500 µL phenol and 500 µL chloroform to the aqueous layer. Centrifuge at max speed for 5 min at room temperature. Pipette aqueous layer into a new 2 mL Eppendorf tube.

SECTION: High Molecular Weight DNA Extraction
35. Repeat step 33-34  until there is no remaining protein debris between the aqueous and the phenol layer.
SECTION: High Molecular Weight DNA Extraction
36. Add 1 mL chloroform to the aqueous layer. Centrifuge at full speed for 5 min. Pipette aqueous layer into a clean 2 mL Eppendorf tube.

SECTION: High Molecular Weight DNA Extraction
37. Repeat step 36 once.
SECTION: High Molecular Weight DNA Extraction
38. Precipitate the DNA by adding 3 M sodium acetate to a final concentration of 0.1 M (~67 µL in 2 mL) and fill to 2 mL with 100% EtOH.

SECTION: High Molecular Weight DNA Extraction
39. Precipitate DNA at -80℃ for at least 120 min or overnight.

SECTION: High Molecular Weight DNA Extraction
40. Centrifuge samples at maximum speed at 4° C for 60 min.

SECTION: High Molecular Weight DNA Extraction
41. Remove supernatant and resuspend DNA pellet in 1 mL 70% EtOH.

SECTION: High Molecular Weight DNA Extraction
42. Centrifuge samples at maximum speed at 4° C for 60 min.

SECTION: High Molecular Weight DNA Extraction
43. Remove supernatant. Allow residual EtOH to evaporate on a heat block set to 37° C.
SECTION: High Molecular Weight DNA Extraction
44. Resuspend DNA pellet in MilliQ water. 
SECTION: Viral Particle Preparation and Lysis
12.1. Do not allow to solidify: hold in a water bath set to 50° C.
SECTION: Viral Particle Preparation and Lysis
15.1. Add 750 µL 20% SDS, 500 µL 0.5 M EDTA, and 200 µL 3 M sodium acetate to a conical tube.

SECTION: Viral Particle Preparation and Lysis
15.2. Bring to 50 mL with MilliQ water.

SECTION: Viral Particle Preparation and Lysis
15.3. Filter sterilize through a 0.22 µM syringe filter.
SECTION: High Molecular Weight DNA Extraction
32.1. If phenol-agarose has frozen, allow sample to thaw before centrifugation.
SECTION: High Molecular Weight DNA Extraction
45. Quantify extracted DNA on Nanodrop or Qubit per manufacturer’s specifications before sequencing.