Extraction of High Molecular Weight DNA from Aureococcus anophagefferens Virus
Alex Truchon, Eric Gann, Steven W Wilhelm
Abstract
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.
Steps
Viral Particle Preparation and Lysis
Infect 1L of Aureococcus anophagefferens CCMP1984 with 10mL of Aureococcus anophagefferens Virus (AaV) lysate.
Incubate cultures at 19 degrees C on a 14:10 light-dark cycle until complete lysis of the culture (~10 days).
Concentrate the lysed culture to a final volume of 50 – 100 mL final volume via tangential flow filtration using a 30 kDa filter.
Centrifuge concentrated lysate for 0h 10m 0s at 3,500 xg at 4° C to pellet cellular organisms in lysate.
Remove supernatant and discard pellet.
Add 10% Triton-X to supernatant to a final concentration of 1%.
Centrifuge 50 mL lysate at a speed of 24,000 xG at 4° C for 1h 15m 0s to pellet viruses. Discard supernatant.
Resuspend the pelleted viruses in a 500µL of 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2.
Add 9µL DNAse I (2.0 mg/mL) and mix gently.
Incubate at room temperature for 1h 0m 0s.
Add 6µL of 500 mM EDTA (pH 8.0). Mix gently.
While incubating, prepare 100mL 2% molecular grade agarose solution by microwaving agarose/water mixture .
Do not allow to solidify: hold in a water bath set to 50° C.
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.
Cast agarose—lysate mixture in BioRad CHEF Mapper Plug Molds (Catalog No. 1703713) and allow to solidify at room temperature.
Prepare viral lysis buffer.
Add 750µL 20% SDS, 500 µL 0.5 M EDTA, and 200 µL 3 M sodium acetate to a conical tube.
Bring to 50mL with MilliQ water.
Filter sterilize through a 0.22 µM syringe filter.
After agarose has solidified punch out of molds and add two per Eppendorf tube with 1mL lysis buffer.
Add proteinase-K to a final concentration of 1 mg/ml to each tube.
Incubate over night at 37° C under gentle rotation (~ 2-3 xG or 100 rpm) in a shaking incubator.
Cast a 2% low-melting agarose gel and allow at least 1h 0m 0s for gel to solidify.
Decant lysis buffer from overnight incubations of plugs.
Prepare TNE wash buffer (10 mM Tris-Base, 200 mM NaCL, 0.5 mM EDTA). Filter sterilize through a 0.22 µM syringe filter.
Wash with 1mL TNE buffer by resuspending and shaking at room temperature for 0h 10m 0s.
Pour off TNE buffer.
Repeat steps 22-23 once.
Carefully insert plugs into individual wells of the low melting agarose gel using a sterile razor blade or pipette tip.
Seal the wells by pouring liquid 2% agarose over the wells and allow poured agarose to solidify.
Run gel at approximately 120 V for 1h 30m 0s to2h 0m 0s or until a high molecular weight band of DNA can be visualized.
High Molecular Weight DNA Extraction
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.
Melt agarose in a water bath set to 80° C Bring volume in tube up to 500µL with MilliQ water. The melted agarose volume should never exceed 500 µL.
Once entirely melted (after at least ten minutes), add1mL basic phenol to the tube and invert to mix.
Place phenol—agarose mixture at -80° C for 0h 10m 0s
Centrifuge the mixture for 0h 5m 0s at maximum speed on a microcentrifuge at room temperature.
If phenol-agarose has frozen, allow sample to thaw before centrifugation.
Carefully remove upper aqueous layer and place in a clean 2mL Eppendorf tube. Discard the lower phenol layer.
Add 500µL phenol and 500µL chloroform to the aqueous layer. Centrifuge at max speed for 0h 5m 0s at room temperature. Pipette aqueous layer into a new 2 mL Eppendorf tube.
Repeat step 33-34 until there is no remaining protein debris between the aqueous and the phenol layer.
Add 1mL chloroform to the aqueous layer. Centrifuge at full speed for 0h 5m 0s. Pipette aqueous layer into a clean 2 mL Eppendorf tube.
Repeat step 36 once.
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 2mL with 100% EtOH.
Precipitate DNA at -80℃ for at least 2h 0m 0s or overnight.
Centrifuge samples at maximum speed at 4° C for 1h 0m 0s.
Remove supernatant and resuspend DNA pellet in 1mL 70% EtOH.
Centrifuge samples at maximum speed at 4° C for 1h 0m 0s.
Remove supernatant. Allow residual EtOH to evaporate on a heat block set to 37° C.
Resuspend DNA pellet in MilliQ water.
Quantify extracted DNA on Nanodrop or Qubit per manufacturer’s specifications before sequencing.
