Large-input CTAB / chloroform clarification and silica maxi-column purification workflow
Cat.No. D316302 / D316303. For plant and fungal DNA extraction using Protocol A or the polyphenol-rich Protocol B route.
Grind plant or fungal tissue to a fine powder under liquid nitrogen using a mortar and pestle. Transfer disrupted material to an appropriately sized tube and allow liquid nitrogen to evaporate without letting the sample thaw.
Protocol A supports up to 3 g wet tissue or 0.6 g dried tissue. Complete powdering and conservative input remain important because clumped tissue lyses poorly and can lower yield or purity.
Add 16 ml Buffer PAL to the disrupted plant or fungal powder and vortex vigorously. Continue vortexing or pipetting until visible clumps are dispersed.
Do not add PVP-40 to Buffer PAL in Protocol A. The manual notes that 2-mercaptoethanol may be added to improve DNA yield, but the key handling point is complete wetting of the large powder mass before lysis.
Incubate the PAL lysate at 65°C for 30 min. Mix 3–5 times during incubation by inverting the tube.
This CTAB-based lysis releases plant total DNA while maintaining conditions that help separate polysaccharides and other plant-matrix components during later clarification.
Add 16 ml chloroform to the lysate, mix well by vortexing for 15 sec, and centrifuge for 10 min at >4,000 × g.
Chloroform or chloroform:isoamyl alcohol (24:1) may be used according to the preparation guidance. The purpose is clarification, not TRIzol-style RNA phase separation.
Transfer 13 ml of the cleared supernatant to a new tube without disturbing the pellet or lower phase.
The target genomic DNA remains in the cleared aqueous supernatant. Avoid carryover of debris, chloroform-rich material or interface contamination.
Add 100 µl RNase A to the supernatant, mix well and incubate at room temperature for 10 min.
The RNase step is included in the standard D3163 workflow before binding-condition adjustment.
Add 13 ml Buffer GWP to the cleared lysate and mix by inverting the tube 6–8 times.
For smaller recovered supernatant volumes, reduce Buffer GWP proportionally. Uniform mixing is required before maxi-column loading.
Insert a HiPure DNA Maxi Column II into a 50 ml collection tube. Load approximately one half of the binding mixture and centrifuge at 3,000–5,000 × g for 5 min at room temperature in a swing-out rotor.
The large binding volume is loaded in two rounds. Keep the column and collection tube orientation stable when discarding flow-through.
Load the remaining mixture onto the same maxi column and centrifuge again for 5 min. Discard the flow-through and reuse the collection tube.
Do not discard the column with the flow-through. The DNA is retained on the silica membrane after binding.
Add 10 ml Buffer GW2 to the column and centrifuge for 3 min at 3,000–5,000 × g. Discard the flow-through and reuse the collection tube.
Confirm that ethanol has been added to Buffer GW2 before use. Poor GW2 preparation is a direct cause of low recovery or poor downstream performance.
Add 10 ml absolute ethanol to the column and centrifuge for 3 min at 3,000–5,000 × g.
This wash helps remove residual salts and contaminants before the longer dry spin.
Discard the flow-through, reuse the collection tube and centrifuge at 3,000–5,000 × g for 10 min at room temperature.
This large-format drying step is important because residual ethanol in the eluate can inhibit PCR, ligation or Southern blot workflows.
Transfer the column to a 50 ml centrifuge tube. Add 0.75–1.0 ml Buffer AE directly onto the membrane, incubate for 5 min at room temperature, and centrifuge for 5 min at 3,000–5,000 × g.
Using 0.5 ml Buffer AE can increase DNA concentration but may reduce total yield.
Repeat the AE elution once, then store the purified plant or fungal DNA at −20°C.
The second elution is shown because it is listed in the manual and improves recovery from the large silica membrane.
Grind plant or fungal tissue to a fine powder under liquid nitrogen and transfer the frozen powder to an appropriately sized tube without allowing the sample to thaw.
Protocol B supports up to 2 g wet tissue or 500 mg dried tissue. Use this route when polyphenol-rich material requires stronger inhibitor control than the standard route.
Add 13 ml Buffer PAL / PVP-40 to the disrupted sample and vortex vigorously. Continue vortexing or pipetting until clumps are removed.
Before use, the manual specifies adding PVP-40 and 2-mercaptoethanol to Buffer PAL to a final concentration of 2%. In practical notation, this corresponds to 2% (w/v) PVP-40 and 2% (v/v) 2-mercaptoethanol. These additives are not provided with the kit.
Incubate the mixture for 30 min at 65°C. Mix 3–5 times during incubation by inverting the tube.
PVP-40 helps bind polyphenolic compounds, while 2-mercaptoethanol supports a reducing environment during large-input plant lysis.
Add 13 ml chloroform to the lysate, mix well, incubate at room temperature for 3 min, and centrifuge for 10 min at >3,000 × g.
This step removes plant debris, proteins, polysaccharides and chloroform-partitioned contaminants. The DNA-containing cleared supernatant is the fraction used for the next step.
Transfer 10 ml of cleared supernatant to a new tube, add 100 µl RNase A, mix well and incubate at room temperature for 20 min.
Do not disturb the pellet or lower phase during transfer. The extended RNase incubation is part of the Protocol B route.
Add 5 ml Buffer GWP to the sample and invert the tube 4–6 times to mix well.
Protocol B uses less GWP than the standard route because the recovered supernatant volume and subsequent ethanol addition differ.
Add 10 ml absolute ethanol and mix thoroughly by vortexing for 15 sec.
A precipitate may form after ethanol addition. Keep the mixture homogeneous before column loading; the precipitate does not invalidate the procedure.
Insert a HiPure DNA Maxi Column II into a 50 ml collection tube. Load approximately one half of the binding mixture and centrifuge at 3,000–5,000 × g for 5 min.
Large-volume loading should be controlled and even; avoid splashing mixture onto the rim of the column.
Load the remaining mixture onto the same maxi column and centrifuge for 5 min. Discard the flow-through and reuse the collection tube.
This repeat loading follows the shared Protocol A column route.
Add 10 ml Buffer GW2 and centrifuge for 3 min at 3,000–5,000 × g. Discard the flow-through.
For colored membranes or inhibitor-rich samples, the troubleshooting guidance allows additional ethanol washing when required.
Add 10 ml absolute ethanol and centrifuge for 3 min at 3,000–5,000 × g.
This wash is retained in the displayed workflow because it is listed as a standard step before the dry spin.
Discard the flow-through and centrifuge the column for 10 min at room temperature.
Extended drying is especially important after large-volume ethanol washes.
Transfer the column to a 50 ml centrifuge tube. Add 0.75–1.0 ml Buffer AE directly to the membrane, incubate for 5 min, and centrifuge for 5 min.
For higher concentration, 0.5 ml AE can be used, with the expected trade-off of lower overall recovery.
Repeat the AE elution once and store purified DNA at −20°C.
The second elution supports total DNA recovery from the maxi-column format.
This workflow separates large-input liquid-nitrogen disruption and CTAB / PAL lysis from the shared chloroform clarification and silica maxi-column purification route. It is intended as a practical companion to the product manual rather than a replacement for the official protocol and covers Cat.No. D316302 and D316303. Protocol A is used for standard plant or fungal samples, while Protocol B adds a PVP-assisted route for polyphenol-rich material. The downstream workflow follows DNA-containing cleared-supernatant recovery, RNase treatment, binding-condition adjustment, two-round maxi-column loading, GW2 / ethanol washing, membrane drying and two-step AE elution.
| Sample route | Displayed preparation estimate | Typical manual workflow time |
|---|---|---|
| Protocol A · Standard Plant / Fungus | 8 min disruption + 30 min PAL lysis | 115–130 min |
| Protocol B · Polyphenol-Rich Samples | 7 min disruption + 30 min PAL / PVP lysis | 130–145 min |
Protocol times stated in the product manual are retained where applicable. Steps without explicit timing are estimated for an experienced operator, including liquid-nitrogen grinding, large-volume reagent handling, tube transfer, centrifuge handling, cleared-supernatant recovery, column loading, filtrate disposal, column repositioning, membrane drying, elution and final tube transfer. Because the main incubation, centrifugation, RNase, drying and elution steps in this D3163 Maxi workflow are largely fixed, the total-time range is kept narrow and is intended to reflect cumulative manual handling differences rather than a long variable incubation step. The main timing variables are large-input grinding quality, clump dispersion in PAL, chloroform phase handling, large-volume supernatant transfer, two-round maxi-column loading and 50 ml tube handling.
D3163 follows the same CTAB / silica logic as the mini-format plant DNA workflow, but the input, reagent volumes, centrifugation format and elution volume are scaled for maxi-column processing. CTAB / PAL lysis releases plant total DNA, chloroform removes proteins, polysaccharides and plant debris, and Buffer GWP with ethanol-compatible conditions promotes binding of DNA to the HiPure membrane. Protocol A does not use PVP-40, while Protocol B uses 2% (w/v) PVP-40 and 2% (v/v) 2-mercaptoethanol in Buffer PAL for polyphenol-rich samples.
The most important control points are keeping the ground sample frozen until PAL contact, avoiding excessive input, fully dispersing clumps before 65°C lysis, recovering the correct DNA-containing cleared supernatant without pellet or chloroform carryover, mixing GWP and ethanol thoroughly, loading the maxi column in two controlled rounds, confirming ethanol addition to GW2, completing the dry spin and applying AE directly to the membrane. Incomplete disruption, overloaded matrix, residual ethanol or wrong-phase carryover are the main risks for low yield, colored eluate or downstream inhibition.