Oct 18, 2021
Extracting high molecular weight DNA from Halichondria panicea (Phylum: Porifera)
- Rasmus Dam Wollenberg1,
- Brian Strehlow2,
- Astrid Schuster3
- 1DNA Sense;
- 2University of Southern Denmark, Department of Biology;
- 3University of Southern Denmark, Department of Biology
External link: https://doi.org/10.1186/s13104-022-06013-3
Protocol Citation: Rasmus Dam Wollenberg, Brian Strehlow, Astrid Schuster 2021. Extracting high molecular weight DNA from Halichondria panicea (Phylum: Porifera). protocols.io https://dx.doi.org/10.17504/protocols.io.yvkfw4w
Manuscript citation:
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: March 05, 2019
Last Modified: October 18, 2021
Protocol Integer ID: 21132
Keywords: DNA extraction, High Molecular Weight DNA, Sponge, Porifera, high molecular weight dna from halichondria panicea, high molecular weight dna from the sponge, extracting high molecular weight dna, halichondria panicea, high molecular weight dna, dna, phylum, molecular biology, porifera, sponge
Abstract
This protocol was used to extract high molecular weight DNA from the sponge (porifera) Halichondria panicea. This protocol contains slight modifications from that presented in Ausubel et al (1995).
References:
Ausubel, F.; Brent, R.; Kingston, R.; Moore, D.; Seidman, J.G.; Smith, J.;Struhl, K. Short Protocols in Molecular Biology(1995), 3rd ed., Unit 2.1: page 2-3
Materials
MATERIALS
Buffer P3Catalog #19053
RNase AQiagenCatalog #19101
Isopropanol
Buffered Phenol Chloroform Isoamyl alcohol (P:C:I) ((25:24:1, saturated with 10 mM Tris, pH 8.0 and 1 mM EDTAMerck MilliporeSigma (Sigma-Aldrich)Catalog #Sigma P2069
70% Ethanol
Proteinase K (2 ml)QiagenCatalog #19131
Buffer AP1QiagenCatalog #1014630
Molecular biology grade 3M sodium-acetate pH 5.2±0.1 Merck MilliporeSigma (Sigma-Aldrich)Catalog #S7899
Buffer AEQiagenCatalog #19077
Note: one could also make up a solution of 800 mM guanidine HCl, 20 mM Tris-Cl pH 8.0, 30 mM EDTA, 5 % Tween-20, and 0.5 % Triton-X 100 to replace Buffer AP1.
Similarly, buffer P3 can be replaced with 3 M potassium acetate buffer at pH 5.5.
AE buffer can be make up with 10 mM Tris-HCl pH 8.0, 1 mM EDTA, and 0.1 % Tween 20.
Rinse sample in filter sterilized seawater three times. Use a mortar and pestle to finely grind approximately 1 gram of tissue in liquid nitrogen.
Transfer homogenized sample into a 15 mL tube.
Add 5 mL of buffer AP1 (QIAGEN, Germany) and mix with 50 µL of 100 mg/mL RNase A. Invert tube a couple of times to mix. Incubate at 65°C for 30-60 minutes, inverting the tube approximately every 5-10 minutes (as often as possible). During this step, cells are lysed in presence of SDS. RNase will remove RNA.
Allow the tube to "cool" to approximately 50°C. Add 150 µL 20 mg/mL Proteinase K. Mix by inverting the tube a couple of times and then incubate at 50°C for a minimum of 2 hours. As often as possible, invert the tube to mix. Proteinase K will digest intracellular proteins such as histones and other DNA-binding proteins. It will also digest membrane-bound proteins which will further facilitate cell-lysis.
Cool the sample on ice for 5-10 minutes (depending on the volume) and add 1,700 µL of P3 (QIAGEN, Germany) buffer. Mix gently by inverting the tube 5-10 times. This step will precipitate SDS and polysaccharides (if present).
Spin down the sample in centrifuge at 7,200 RCF for 30 minutes.
Transfer supernatant with a wide-bore, i.e. cut with sterile blade, P1000/P5000 pipette-tip to a 15 mL tube. Subsequently, add Chloroform:Phenol:Isoamyl in a 1:1 mixture by volume. For initial protocol evaluation, vortex the sample until emulsions have formed. Prolonged incubation is encouraged. If downstream DNA yields turn out high, revert to a non-vortexing approach to minimize DNA shearing (gentle, prolonged mixing) Prolonged incubation is encouraged/essential.
Spin down the sample at 7,200 RCF for 20 minutes and remove the DNA-containing supernatant (slowly and wide a wide-bore tip).
Repeat step 7-8 one to two times and subsequently transfer the supernatant to a clean 15 mL tube.
Add Molecular biology grade 3M sodium-acetate pH 5.2±0.1 (S7899 Sigma) to a final concentration of 0.3 M. This step will lower pH and neutralize the phosphodiester backbone, thus rendering the DNA ready for precipitation.
Add 1 volume of room-temperature isopropanol if HMW DNA is the intended target or use 1 volume of ice-cold isopropanol if both LMW and HMW is the target (with the option of including an over-night incubation at -20°C). For HMW DNA, invert tube a few times to mix until DNA precipitates as nice jelly-fish threads (within a few min). Then centrifuge for 10 min at 7,200 RCF (at room-temperature if HMW DNA is the target).The short incubation with isopropanol will preferentially precipitate HMW DNA species and by keeping the sample at room-temperature, salt carry-over is further minimized.
Remove the supernatant and add as much ice-cold 70 % ethanol as possible and gently invert tube to mix. Gently dislodge (if possible) the pellet. Centrifuge for 10 min at 7,200 RCF and replace as much liquid with fresh ice-cold 70 % ethanol. Spin again and remove as much liquid as possible. Finally aspirate residual ethanol with a P10 pipette (after a short spin).This will remove salts and replace isopropanol with the more volatile ethanol.
Air-dry the pellet for 5-10 minutes. Do not over-dry!
Add 100-200 µL AE buffer. Incubate the sample at room-temperature or fridge for a minimum of two days. Longer incubations are encouraged (1-2 weeks if time allows and no DNase activity is assumed present). If the pellet is not readily dissolving, then try to pull out "DNA threads" with a P10 pipette.
Measure quality and concentration with NanoDrop (or similar) and dsDNA (Qubit) and evaluate DNA size-distribution.
If A260/A230 is < 2.0, perform a clean-up step with the Qiagen PowerPro CleanUp kit.
Measure quality and concentration with NanoDrop (or similar) and dsDNA (Qubit) and evaluate DNA size-distribution.