TITLE: High-molecular weight DNA extraction from challenging fungi using CTAB for lysis and precipitation
AUTHORS: Ashley Jones, Benjamin Schwessinger

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left"><span>Extracting pure high-molecular weight DNA from fungi is often difficult due to the presence of polysaccharides and potentially other compounds, which biochemically mimic DNA or interfere with the DNA extraction process. Such compounds can co-elute with DNA in many extraction methods, making them difficult to remove from solution. Some contaminants may even be undetected by spectrophotometers or fluorometric devices, however they still substantially interferes with long-read DNA sequencing. In attempt to resolve these challenges, a protocol is presented that utilses cetrimonium bromide (CTAB) for lysis and precipitation. CTAB in the lysis solution acts as a cationic detergent to dissolve cell membranes and remove polysaccharides by the formation of CTAB-polysaccharide complexes that co-precipitate during chloroform phase separation. Using CTAB for DNA precipitation under low salt, low ionic conditions, CTAB-DNA complexes form while leaving polysaccharides in solution, resulting in significantly less co-precipitation than alcohol methods. The presented protocol also includes some updates to current strategies and incorporates two different options for DNA clean-up and size selection. Using this protocol, we have been successfully sequencing various fungi with a MinION (Oxford Nanopore Technologies). For wheat stripe rust </span><span style = "font-style:italic;">Puccinia striiformis</span><span> and wheat leaf rust </span><span style = "font-style:italic;">Puccinia triticina, </span><span>we performed the presented method followed by size selection with an automated gel purification system, with sequencing yielding 5.88 Gbp with an N50 of 34.79 kb and 4.88 Gbp with an N50 of 27.61 kb, respectively for the two fungi. These fungi are notorious for being recalcitrant and these results have been positive improvements on many other methods. To increase sequencing output for these samples, more work is needed to identify and remove the elusive contaminants. Other fungi, such as sorghum rot fungus </span><span style = "font-style:italic;">Macrophomina phaseolina</span><span>, we chose an alternative size selection method, precipitation with a polymer and salt solution. This sequencing yielded 13.71 Gbp with an N50 of 21.75 kb, while another strain yielded 9.72 Gbp with an N50 of 43.50 kb. </span></div></div></div>

[STEPS]
?. [PREPARATION]
Grow fungus and collect ~500 mg of sample (spores for rust fungi). Keep frozen at -80°C or liquid nitrogen until ready.
Ideal for fungi that have a small genome, approximately 50 Mb.Take into consideration cell density and genome size; low cell density and/or smaller genomes may need to be prepped twice, larger genomes may not need many spores.
?. [PREPARATION]
Set a rotating/shaking incubator (e.g. hybridisation oven) or alternatively a water bath to 60°C. This will be used to dissolve CTAB solutions and later to preciptate DNA with CTAB (best results if rotating or gently shaking).
DNA precipitation with CTAB will be optimal if rotating or gently shaking. A water bath will still be suitable, but can produce a lower DNA yield due to a lack of CTAB-DNA complexes forming. Temperature was originally 55°C, 60°C appears to give better results and not shear the DNA any further. This may beneficial to both lysis and precipitation steps, but has not been extensively tested.
?. [PREPARATION]
Freshly prepare lysis and precipitation buffers.Lysis buffer (pH 8)15 mL of lysis buffer per sample (~500 mg). Prepare in a 50 mL Falcon tubeBefore adding NaOH: pH 7.4. After adding NaOH: pH 8.  Precipitation buffer (pH 8)30 mL of lysis buffer per sample (~500 mg).Prepare in a 50 mL Falcon tubeBefore adding NaOH: pH 7.6. After adding NaOH: pH 8.Reagent Target concentrationMolecular weightStock concentrationFrom stockCTAB4% (w/v)364.45powder0.60 gTris-HCl (pH 8)100 mM157.601 M1.50 mLEDTA (pH 8)20 mM292.240.5 M0.60 mLNaCl1.2 M58.445 M3.60 mLPVP-401%40,00010%1.50 mLMilli-Q water---~6.95 mLNaOHAdjust pH to 8 40.001 M~0.25 mL----Total 15 mLPrecipitation buffer (pH 8)30 mL of lysis buffer per sample (~500 mg).Prepare in a 50 mL Falcon tubeBefore adding NaOH: pH 7.6. After adding NaOH: pH 8.ReagentTarget concentrationMolecular weightStock concentrationFrom stockCTAB2% (w/v)364.45powder0.60 gTris-HCl (pH 8)100 mM157.601 M3 mLEDTA (pH 8)20 mM292.240.5 M1.20 mLMilli-Q water---~24.90 mLNaOHAdjust pH to 840.001 M~0.30 mL----Total 30 mL
Reagent Target concentrationMolecular weightStock concentrationFrom stockCTAB4% (w/v)364.45powder0.60 gTris-HCl (pH 8)100 mM157.601 M1.50 mLEDTA (pH 8)20 mM292.240.5 M0.60 mLNaCl1.2 M58.445 M3.60 mLPVP-401%40,00010%1.50 mLMilli-Q water---~6.95 mLNaOHAdjust pH to 8 40.001 M~0.25 mL----Total 15 mL
ReagentTarget concentrationMolecular weightStock concentrationFrom stockCTAB2% (w/v)364.45powder0.60 gTris-HCl (pH 8)100 mM157.601 M3 mLEDTA (pH 8)20 mM292.240.5 M1.20 mLMilli-Q water---~24.90 mLNaOHAdjust pH to 840.001 M~0.30 mL----Total 30 mL
The precipitation buffer contains no NaCl. When 2x volume is added to the sample at later stages, the NaCl in the initial lysis buffer is diluted from 1.2 M to ≤ 0.4 M. This triggers DNA precipitation by the formation of CTAB-DNA nucleation complexesCTAB was originally 2% in the lysis buffer. Increasing CTAB to 4% in lysis buffer appears to have beneficial effects on DNA yield and no negative consequence have been observed. Originally, pH was not adjusted to pH 8 in either lysis or precipitation buffers. Adjusting pH to 8 appears to have beneficial effects on DNA yield and no negative consequence have been observed. The DNA extraction process still worked when the lysis and precipitation buffers were not adjusted to pH 8. Note Tris-HCl and EDTA stock solutions are already at pH 8.Original publication (Arseneau et al., 2017) used 500 μL of lysis buffer per 25 mg of mollusc tissue. Amount of lysis buffer per sample could be scaled down.EDTA is a metal ion chelator. By binding to Mg2+, DNase activity is stopped as it is dependent on Mg2+. CTAB was originally 2% in the lysis buffer. Increasing CTAB to 4% in lysis buffer appears to have beneficial effects on DNA yield and no negative consequence have been observed. Originally, pH was not adjusted to pH 8 in either lysis or precipitation buffers. Adjusting pH to 8 appears to have beneficial effects on DNA yield and no negative consequence have been observed. The DNA extraction process still worked when the lysis and precipitation buffers were not adjusted to pH 8. Note Tris-HCl and EDTA stock solutions are already at pH 8.Original publication (Arseneau et al., 2017) used 500 μL of lysis buffer per 25 mg of mollusc tissue. Amount of lysis buffer per sample could be scaled down.EDTA is a metal ion chelator. By binding to Mg2+, DNase activity is stopped as it is dependent on Mg2+. Originally, pH was not adjusted to pH 8 in either lysis or precipitation buffers. Adjusting pH to 8 appears to have beneficial effects on DNA yield and no negative consequence have been observed. The DNA extraction process still worked when the lysis and precipitation buffers were not adjusted to pH 8. Note Tris-HCl and EDTA stock solutions are already at pH 8.Original publication (Arseneau et al., 2017) used 500 μL of lysis buffer per 25 mg of mollusc tissue. Amount of lysis buffer per sample could be scaled down.EDTA is a metal ion chelator. By binding to Mg2+, DNase activity is stopped as it is dependent on Mg2+. The DNA extraction process still worked when the lysis and precipitation buffers were not adjusted to pH 8. Note Tris-HCl and EDTA stock solutions are already at pH 8.Original publication (Arseneau et al., 2017) used 500 μL of lysis buffer per 25 mg of mollusc tissue. Amount of lysis buffer per sample could be scaled down.EDTA is a metal ion chelator. By binding to Mg2+, DNase activity is stopped as it is dependent on Mg2+. Original publication (Arseneau et al., 2017) used 500 μL of lysis buffer per 25 mg of mollusc tissue. Amount of lysis buffer per sample could be scaled down.EDTA is a metal ion chelator. By binding to Mg2+, DNase activity is stopped as it is dependent on Mg2+.
?. [PREPARATION]
Vortex lysis and precipitation buffers, then place both in the 60°C incubator or water bath. Leave buffers at 60°C until needed at later steps.
Necessary to dissolve the CTAB and may take 20 min or longer. Further vortexing or inverting may be also be required.
?. [SAMPLE LYSIS]
Place a sterile mortar and pestle into an insulated container. Pour liquid nitrogen into the container to chill the mortar and pestle.
The lid of an ice box can be used. Ensure the container will not crack and spill liquid nitrogen.
?. [SAMPLE LYSIS]
Add sample to the mortar and grind throughly (approximately 2-3 min). Keep the mortar and pestle chilled by having liquid nitrogen within the surrounding container.
Avoid pouring liquid nitrogen directly into the mortar or onto the sample, as the sample will disperse all over the pestle and potentially the table.
?. [SAMPLE LYSIS]
Transfer the ground sample and sand to a tube of lysis buffer.
?. [SAMPLE LYSIS]
Add RNAse A and Proteinase K to the lysis buffer:EnzymeTarget concentrationStock concentrationFrom stockRNAse A200 μg/mL20 mg/mL150 μLProteinase K200 μg/mL20 m/mL150 μL
EnzymeTarget concentrationStock concentrationFrom stockRNAse A200 μg/mL20 mg/mL150 μLProteinase K200 μg/mL20 m/mL150 μL
RNAse A was added earlier than Arseneau et al. (2017) and concentration increased from 50 μg/mL to 200 μg/mL, which is more suitable when adding to a lysis buffer opposed to purified DNA. With less RNAse A, RNA was still present after extraction. Athough RNAse A concentration is generally recommended to be 1-100 μg/mL, Qiagen DNeasy plant kits use as much as 2,000 μg/mL. RNAse T has less DNase activity (still some), but was less effective at degrading fungal RNA and more costly.Arseneau et al. (2017) included Proteinase K at a final concentration of 1,130 μg/mL to break down mollusc tissue. Concentrations this high have not been tested with fungi.RNAse A is still active in EDTA as it is not Mg2+ dependent and doesn't appear to require other metal ions as cofactors. Similarily, Proteinase K is still active, as the two binding sites for Ca2+ are not directly involved in the proteolysis catalytic mechanism.RNAse A and Proteinase K can co-exist in the same solution. RNAse A is highly resistant to proteolysis by Proteinase K. Both are recommended to be 50-100 µg/mL. RNAse A is active at temperatures15-70°C (optimal at 60°C), pH 6-10 (optimal pH 7.6). Proteinase K is active at temperatures 20-60°C (optimal 50-60°C), pH 4-12 (optimal pH 8).Arseneau et al. (2017) included Proteinase K at a final concentration of 1,130 μg/mL to break down mollusc tissue. Concentrations this high have not been tested with fungi.RNAse A is still active in EDTA as it is not Mg2+ dependent and doesn't appear to require other metal ions as cofactors. Similarily, Proteinase K is still active, as the two binding sites for Ca2+ are not directly involved in the proteolysis catalytic mechanism.RNAse A and Proteinase K can co-exist in the same solution. RNAse A is highly resistant to proteolysis by Proteinase K. Both are recommended to be 50-100 µg/mL. RNAse A is active at temperatures15-70°C (optimal at 60°C), pH 6-10 (optimal pH 7.6). Proteinase K is active at temperatures 20-60°C (optimal 50-60°C), pH 4-12 (optimal pH 8).RNAse A is still active in EDTA as it is not Mg2+ dependent and doesn't appear to require other metal ions as cofactors. Similarily, Proteinase K is still active, as the two binding sites for Ca2+ are not directly involved in the proteolysis catalytic mechanism.RNAse A and Proteinase K can co-exist in the same solution. RNAse A is highly resistant to proteolysis by Proteinase K. Both are recommended to be 50-100 µg/mL. RNAse A is active at temperatures15-70°C (optimal at 60°C), pH 6-10 (optimal pH 7.6). Proteinase K is active at temperatures 20-60°C (optimal 50-60°C), pH 4-12 (optimal pH 8).
?. [SAMPLE LYSIS]
Mix thoroughly by vortexing and inverting the solution. Incubate at 60°C for 60-90 min
Ideal to be rotating or gently shaking, but this is not as critical as CTAB-DNA precipitation at later steps.
?. [CHLOROFORM CLEAN-UP]
Sample should be in a 50 mL Falcon tube, if not and the sample with lysis buffer occupies more than 50% of the tube capacity, move to a bigger tube or split the sample evenly across two appropriately sized tubes.
?. [CHLOROFORM CLEAN-UP]
Add an equal volume of chloroform: isoamyl alcohol (24:1, v/v) and mix by inverting 10-15 times.
Ensure the organic and aqueous phases become mixed at least temporarily.Solution may appear opaque, but this is not a concern.
?. [CHLOROFORM CLEAN-UP]
Separate the phases by centrifuging at 5,000 rcf for 10 min at 20°C (or room temperature).
Chloroform is denser than water, it will mix with the oragnic phase which settles to the bottom and the aqueous phase is at the top. Some protocols recommend centrifuging at 12,000 rpm. Not tested, however if centrifugation hasn't been sufficient the interphase will be cloud-like and poorly compacted, with oraganic substances still in the aqueous phase.
?. [CHLOROFORM CLEAN-UP]
Repeat the chloroform: isoamyl alcohol clean (equal volume).
?. [DNA PRECIPITATION]
Add 2 volumes of precipitation buffer (30 mL to ~15 mL chloroform cleaned sample). Mix by swirling and inverting the tube several times.
?. [DNA PRECIPITATION]
Centrifuge at 16,000 rcf for 20 minutes at 20°C (or room temperature) to pellet crystals.
Was originally 10 min, Arseneau et al. (2017) does 3 min. More pellet has been observed the longer the centrifugation. Centrifuging 2x 15 min, the second spin collected another 8% yield. Extensive centrifugation at 16,000 rcf could lead to DNA shearing. However 30 mins appears fine with most DNA being 20-50 kb, with a tail of ultra long fragments up to 200 kb. Centrifuging 2x 15 min, the second spin collected another 8% yield. Extensive centrifugation at 16,000 rcf could lead to DNA shearing. However 30 mins appears fine with most DNA being 20-50 kb, with a tail of ultra long fragments up to 200 kb.
?. [DNA PRECIPITATION]
Carefully decant the supernatant as soon as possible, without disturbing the pellet. Care must be taken as the pellet is fragile, being easily dislodged.
It is good practice to save the supernatent in another tube, in case the DNA has not precipitated or the pellet has been unknowingly dislodged.
?. [DNA PRECIPITATION]
Add approximately 10-15 mL freshly prepared 80% ethanol, enough to cover the pellet. Gently swirl and invert the tube.
?. [DNA PRECIPITATION]
Centrifuge at 16,000 rcf for 5 min.
?. [DNA PRECIPITATION]
Air-dry the pellet for 10-15 min, or until all ethanol has evaporated.
?. [DNA PRECIPITATION]
Pipette 100-200 µL of 10 mM Tris-HCl pH 8 (or nuclease-free water) onto the DNA pellet. Repeatedly pipette the same 100-200 µL onto the pellet until it disloges from the tube wall and dissolves. Proceed with care to avoid pipette mixing as much as possible, to prevent DNA shearing.
Elution volume is based on pellet size and how the sample will be size selected later. Gentle flicking of the tube is ideal but not possible here if using 50 mL Falcon tube.Wide-bore pipette tips are also an option.Gentle flicking of the tube is ideal but not possible here if using 50 mL Falcon tube.Wide-bore pipette tips are also an option.
?. [DNA PRECIPITATION]
Transfer DNA to a 1.5 mL DNA LoBind Eppendorf tube.
?. [DNA PRECIPITATION]
Quantify the DNA on a Nanodrop and a Qubit fluorometer (dsDNA broad-range assay) (both instruments from Thermo Fisher Scientific). The DNA will be somewhat crude, containing some degraded RNA and potentially residual protein or other contaminants, which will be removed later.
For Nanodrop, use 1 μL. As degraded RNA may still be present, Nanodrop results may give a large over-estimation of concentration.Qubit fluorometer is the most reliable. However, it is highly dependent on the accuracy of the amount pipetted. Use 2 μL when sample is plentiful. Using 1 μL is prone to pipetting errors.For pure DNA, Nanodrop:Qubit is 1:1, 260/280 is 1.8 and 260/230 is 2.0.Qubit fluorometer is the most reliable. However, it is highly dependent on the accuracy of the amount pipetted. Use 2 μL when sample is plentiful. Using 1 μL is prone to pipetting errors.For pure DNA, Nanodrop:Qubit is 1:1, 260/280 is 1.8 and 260/230 is 2.0.
?. [DNA PRECIPITATION]
Store DNA at 4°C to prevent cycles of freeze-thawing that shear the DNA.
No effects on DNA integrity have been noticed for samples stored at 4°C for extended periods.
?. [DNA CLEAN-UP AND SIZE SELECTION OPTION 2: GEL PURIFICATION BY PIPPIN PREP]
Using a PippinHT (Sage Science) or similar automated electrophoresis system, gel purify 20-30 µg, following the manufacturer’s instructions. A 15 kb high pass separation is recommended, however if DNA is plentiful, 20 kb high pass is more suitable. The PippinHT has 12 lanes, however a lane pair needs be dedicated to an external ladder, leaving 10 lanes for samples. 20 µL of DNA goes into each lane with 5 µL of loading buffer (200 µL of total sample). The manufacturer recommends a maximum 1.5 µg per lane (15 µg total per cassette), however, can be successfully overloaded to 3 µg per lane (perhaps more). The size selection will still be very accurate.
?. [DNA CLEAN-UP AND SIZE SELECTION OPTION 2: GEL PURIFICATION BY PIPPIN PREP]
After separation, wait at least 45 min (hours or overnight is suitable), to aid elution and recovery.
?. [DNA CLEAN-UP AND SIZE SELECTION OPTION 2: GEL PURIFICATION BY PIPPIN PREP]
Collect the contents of all elution wells into a 2 mL or 1.5 mL Eppendorf tube (~250-300 µL).
?. [DNA CLEAN-UP AND SIZE SELECTION OPTION 2: GEL PURIFICATION BY PIPPIN PREP]
Add 30 µL of 0.1% tween in electrophoresis buffer to each elution well (provided in kit). Wait for 5 min and then transfer the contents to the same Eppendorf tube (additional ~300 µL, tube total is ~600 µL).
?. [DNA CLEAN-UP AND SIZE SELECTION OPTION 2: GEL PURIFICATION BY PIPPIN PREP]
Add at least 1x volume of AMPure XP beads (~600 µL). Incubate at room temperature for 5 min.
If using 2% Sera-Mag beads in the lab, alternatively fill ~90% of the tube capacity with binding buffer (will be > 2x volume for 2 mL tubes) and add 100 µL of 2% Sera-Mag beads.
?. [DNA CLEAN-UP AND SIZE SELECTION OPTION 2: GEL PURIFICATION BY PIPPIN PREP]
Place on a magnetic rack for 5 min, or until the solution becomes clear. Discard the supernatant.
?. [DNA CLEAN-UP AND SIZE SELECTION OPTION 2: GEL PURIFICATION BY PIPPIN PREP]
Keeping the tube on the magnetic rack, add 1 mL of freshly prepared 80% ethanol. Discard the ethanol and repeat for a second ethanol wash.
?. [DNA CLEAN-UP AND SIZE SELECTION OPTION 2: GEL PURIFICATION BY PIPPIN PREP]
Remove all traces of ethanol and let the beads air dry for 1-4 min.
Important! Do not let the beads dry completely, they will crack and significantly reduce DNA recovery.
?. [DNA CLEAN-UP AND SIZE SELECTION OPTION 2: GEL PURIFICATION BY PIPPIN PREP]
Remove the tube from the magnetic rack and elute with 50 µL of 10 mM Tris-HCl pH 8 (or nuclease-free water). Incubate for 10 min at room temperature.
A maximum DNA volume of 48 µL is used in an Oxford Nanopore ligation preparation.
?. [DNA CLEAN-UP AND SIZE SELECTION OPTION 2: GEL PURIFICATION BY PIPPIN PREP]
Place on a magnetic rack for 5 min, or until the solution becomes clear. Transfer eluted DNA to a 1.5 mL DNA LoBind Eppendorf tube.
?. [DNA CLEAN-UP AND SIZE SELECTION OPTION 2: GEL PURIFICATION BY PIPPIN PREP]
Perform an additional, second elution on the beads, remove from magnet, resuspend (another 50 µL), incubate, magnetise and transfer to another 1.5 mL DNA LoBind Eppendorf tube.
Save this second elution for other applications such as running a pulse field gel or creating and Illumina short-read library.
?. [DNA CLEAN-UP AND SIZE SELECTION OPTION 2: GEL PURIFICATION BY PIPPIN PREP]
Quantify the DNA on a Nanodrop and a Qubit fluorometer (dsDNA broad-range assay) (both instruments from Thermo Fisher Scientific). The DNA should be pure, free of contaminants.
Expect 10-30% recovery relative to total input.
?. [DNA CLEAN-UP AND SIZE SELECTION OPTION 2: GEL PURIFICATION BY PIPPIN PREP]
Store DNA at 4°C to prevent cycles of freeze-thawing that shear the DNA.
No effects on DNA integrity have been noticed for samples stored at 4°C for extended periods.
?. [DNA CLEAN-UP AND SIZE SELECTION OPTION 1: PRECIPITATION WITH POLYMERS]
Utilise a polymer and salt solution listed in the following protocol (or commercial option such as Short-Read Elimator kit from Circulomics). The size selection section is all that is required.  DNA clean-up and size selection for long-read sequencing v4Jones, A., Purushotham, N., Nasim, J., and Schwessinger, B. (2021). DNA clean-up and size selection for long-read sequencing. Protocols.io v4, dx.doi.org/10.17504/protocols.io.bwkdpcs6.
?. EXPECTED RESULTSUsing the protocol described, we performed DNA extractions for wheat stripe rust Puccinia striiformis and wheat leaf rust Puccinia triticina, followed by size selection with an automated gel purification system (PippinHT). We have been obtaining clean high-molecular weight DNA (Table 1, Figure 1). Nuclear DNA fragment sizes appear above 10 kb on agarose gels, with mitochondrial DNA and degraded RNA still present until size selection was performed (Figure 2). For sequencing, we adopted the portable MinION sequencer from Oxford Nanopore Technologies. During sequencing, we can obtain 5.88 Gbp with an N50 of 34.79 kb and 4.88 Gbp with an N50 of 27.61 kb, respectively for the two fungi (Table 2, Figure 3). This includes quality reads over 100 kb in length (> Q7, Phred scale). These fungi are notorious for being recalcitrant and these results have been positive improvements on many other methods. To increase sequencing output for these samples, more work is needed to identify and remove the elusive contaminants that are negatively affecting sequencing.  Other fungi, such as sorghum rot fungus Macrophomina phaseolina, we chose an alternative size selection method, precipitation with a polymer and salt solution. This sequencing yielded 13.71 Gbp with an N50 of 21.75 kb, while another strain yielded 9.72 Gbp with an N50 of 43.50 kb. These results can be found in the relevant DNA clean-up protocol mentioned. A troubleshooting guide for MinION sequencing is presented in Figure 4, illustrating the most common problems researchers have.
?. [PREPARATION]
Collect liquid nitrogen and the sample. Place sample in liquid nitrogen until ready.
?. [SAMPLE LYSIS]
Add approximately 1 g of acid-washed autoclaved sand to the mortar.
Can be increased to 2 g if necessary to help grind the sample or add volume.
?. [CHLOROFORM CLEAN-UP]
Transfer the upper aqueous phase to a new 50 mL Falcon tube.
?. [DNA PRECIPITATION]
Incubate at 60°C in a rotating/shaking incubator for 2.5 h or until white crystals of CTAB-DNA complex can be observed floating inside the tubes (incubate at least 1 h).
This step is critical for DNA precipitation, as CTAB will nucleate around the DNA slowly and works best with movement (e.g. 150 rpm). Increasing the speed helps.It is very difficult to see any crystals and likely depends on the sample. For samples with large yields over 77 μg, I did not observe crystals.No further yield was seen incubating longer than 2.5 h (including overnight incubation). The ideal time may be 1.5-2 h but this needs further testing. The extended incubation time does not appear to shear the DNA compared to shorter incubation times that result in significantly lower yields.Precipitation may also be influenced by DNA concentration, which has not been tested. This would require reducing the lysis buffer voume or increasing sample input.As the lysis buffer has now increased CTAB from 2% to 4% (w/v), it is anticipiated this is also helping the precipitation, but has not been extensively tested to confirm this hypothesis. It is very difficult to see any crystals and likely depends on the sample. For samples with large yields over 77 μg, I did not observe crystals.No further yield was seen incubating longer than 2.5 h (including overnight incubation). The ideal time may be 1.5-2 h but this needs further testing. The extended incubation time does not appear to shear the DNA compared to shorter incubation times that result in significantly lower yields.Precipitation may also be influenced by DNA concentration, which has not been tested. This would require reducing the lysis buffer voume or increasing sample input.As the lysis buffer has now increased CTAB from 2% to 4% (w/v), it is anticipiated this is also helping the precipitation, but has not been extensively tested to confirm this hypothesis. No further yield was seen incubating longer than 2.5 h (including overnight incubation). The ideal time may be 1.5-2 h but this needs further testing. The extended incubation time does not appear to shear the DNA compared to shorter incubation times that result in significantly lower yields.Precipitation may also be influenced by DNA concentration, which has not been tested. This would require reducing the lysis buffer voume or increasing sample input.As the lysis buffer has now increased CTAB from 2% to 4% (w/v), it is anticipiated this is also helping the precipitation, but has not been extensively tested to confirm this hypothesis. The extended incubation time does not appear to shear the DNA compared to shorter incubation times that result in significantly lower yields.Precipitation may also be influenced by DNA concentration, which has not been tested. This would require reducing the lysis buffer voume or increasing sample input.As the lysis buffer has now increased CTAB from 2% to 4% (w/v), it is anticipiated this is also helping the precipitation, but has not been extensively tested to confirm this hypothesis. Precipitation may also be influenced by DNA concentration, which has not been tested. This would require reducing the lysis buffer voume or increasing sample input.As the lysis buffer has now increased CTAB from 2% to 4% (w/v), it is anticipiated this is also helping the precipitation, but has not been extensively tested to confirm this hypothesis.
?. [DNA PRECIPITATION]
Let the pellet soak for 15 min at room temperature to dissolve excess salts and CTAB.
?. [DNA PRECIPITATION]
Carefully decant the supernatant as soon as possible, without disturbing the pellet. Care must be taken as the pellet is fragile, being easily dislodged.
?. [CHLOROFORM CLEAN-UP]
Briefly place at room temperature or on ice, to bring the temperature down.
?. [DNA PRECIPITATION]
Incubate DNA at room temperature for 5-10 min to ensure the pellet has completely hydrated then place on ice.
?. [DNA CLEAN-UP AND SIZE SELECTION OPTION 1: PRECIPITATION WITH POLYMERS]
If the DNA is suspected to have a large amount of degraded RNA, first perform a bead clean with AMPure XP magnetic beads (or equivalent). Use 1x volume of beads and follow the manufacturer's instructions (example bead clean workflow is presented in next section).
Removing many small degraded fragments will increase the performance of the following more stringent size selection.
?. [DNA CLEAN-UP AND SIZE SELECTION OPTION 1: PRECIPITATION WITH POLYMERS]
If some residual degraded RNA is still present, this can be ignored as it will be removed during bead clean-ups during the sequencing library preparation.