Mar 09, 2022
Extraction of High Molecular Weight DNA from Aureococcus anophagefferens Virus
- 1University of Tennessee, Knoxville;
- 2The University of Tennessee, Knoxville
Protocol Citation: Alex Truchon, Eric Gann, Steven W Wilhelm 2022. Extraction of High Molecular Weight DNA from Aureococcus anophagefferens Virus. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjx7owlk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: September 27, 2021
Last Modified: March 09, 2022
Protocol Integer ID: 53587
Keywords: Nucleocytoviricota, virus, DNA extraction, high molecular weight, long-read sequencing
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.
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.
Materials
30 kDA Pelicon Filter
Tangential Flow Filtration Device
10% Triton-X
Tris-HCl
DNAse I
Proteinase K
500 mM EDTA
Molecular grade agarose
Low-melting temperature agarose
BioRad CHEF Mapper Plug Molds (Catalog No. 1703713)
Safety warnings
While working with phenol and chloroform, ensure that all skin is properly covered with the appropriate lab safety attire. Any work with these chemicals should be performed in a chemical fume hood as to avoid inhalation.
Viral Particle Preparation and Lysis
Viral Particle Preparation and Lysis
Infect 1 L of Aureococcus anophagefferens CCMP1984 with 10 mL 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).
1w 3d
Concentrate the lysed culture to a final volume of 50 – 100 mL final volume via tangential flow filtration using a 30 kDa filter.
3h
Centrifuge concentrated lysate for 00:10:00 at 3,500 xg at 4° C to pellet cellular organisms in lysate.
10m
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 01:15:00 to pellet viruses. Discard supernatant.
1h 15m
Resuspend the pelleted viruses in a 500 µL of 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2.
Add 9 µL DNAse I (2.0 mg/mL) and mix gently.
Incubate at room temperature for 01:00:00 .
1h
Add 6 µL of 500 mM EDTA (pH 8.0). Mix gently.
While incubating, prepare 100 mL 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 750 µL 20% SDS, 500 µL 0.5 M EDTA, and 200 µL 3 M sodium acetate to a conical tube.
Bring to 50 mL 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 1 mL 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 01:00:00 for gel to solidify.
1h
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 1 mL TNE buffer by resuspending and shaking at room temperature for 00:10:00 .
10m
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 01:30:00 to02:00:00 or until a high molecular weight band of DNA can be visualized.
3h 30m
High Molecular Weight DNA Extraction
High Molecular Weight DNA Extraction
4h 40m
4h 40m
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 500 µL with MilliQ water. The melted agarose volume should never exceed 500 µL.
Once entirely melted (after at least ten minutes), add1 mL basic phenol to the tube and invert to mix.
10m
Place phenol—agarose mixture at -80° C for 00:10:00
10m
Centrifuge the mixture for 00:05:00 at maximum speed on a microcentrifuge at room temperature.
5m
If phenol-agarose has frozen, allow sample to thaw before centrifugation.
Carefully remove upper aqueous layer and place in a clean 2 mL Eppendorf tube. Discard the lower phenol layer.
Add 500 µL phenol and 500 µL chloroform to the aqueous layer. Centrifuge at max speed for 00:05:00 at room temperature. Pipette aqueous layer into a new 2 mL Eppendorf tube.
5m
Repeat step 33-34 until there is no remaining protein debris between the aqueous and the phenol layer.
Add 1 mL chloroform to the aqueous layer. Centrifuge at full speed for 00:05:00 . Pipette aqueous layer into a clean 2 mL Eppendorf tube.
5m
Repeat step 36 once.
5m
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.
Precipitate DNA at -80℃ for at least 02:00:00 or overnight.
2h
Centrifuge samples at maximum speed at 4° C for 01:00:00 .
1h
Remove supernatant and resuspend DNA pellet in 1 mL 70% EtOH.
Centrifuge samples at maximum speed at 4° C for 01:00:00 .
1h
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.