CTAB / PAL Lysis · Chloroform Clarification · Silica Column Genomic DNA Purification
Cat. No. D3187 · Manual workflow overview for plant and fungal genomic DNA extraction
Grind plant tissue or fungus to a fine powder under liquid nitrogen. Transfer 50–150 mg fresh / frozen sample or 15–40 mg dry sample into a 2 ml centrifuge tube.
For first use, start with about 50 mg fresh sample or 15 mg dry sample. Mucilage-rich samples should usually be reduced to about 30–50 mg fresh material to avoid column overload and viscous lysate.
Add 700 µl Buffer PAL preheated to 65°C immediately, vortex vigorously to disperse the powder, and incubate at 65°C for 20 min. Invert the tube 2–3 times during incubation.
For complex samples, add 2-mercaptoethanol to Buffer PAL at 0.1% (v/v) to improve oxidation resistance; for extremely hard-to-lyse samples, the manual allows 2% (v/v). For simple crop samples such as rice, corn or tomato, 2-mercaptoethanol is optional. The main control point is fast wetting of the powder before local drying or oxidation occurs.
Add 700 µl chloroform, vortex for 15 sec, and centrifuge at 12,000 × g for 5 min at room temperature.
After centrifugation, the usable fraction is the cleared upper aqueous phase. Avoid disturbing the interphase or organic phase.
Carefully transfer the cleared supernatant to a new centrifuge tube.
If RNA-free genomic DNA is required, the optional RNase A treatment is performed here and adds about 15 min; it is not included in the displayed standard timeline.
Add 700 µl Buffer GWP to the recovered supernatant and invert the tube 10–15 times to mix thoroughly.
Uniform mixing is required before column loading, especially when precipitate or viscosity is present.
Insert a HiPure gDNA Mini Column II into a 2 ml collection tube. Load up to 750 µl of the mixture, including any precipitate that may have formed, and centrifuge for 1 min at ≥10,000 × g. Discard the flow-through.
Do not exceed the practical loading capacity of the column. If the mixture contains visible precipitate, keep it dispersed before pipetting.
Load the remaining mixture onto the same column and centrifuge for 1 min. Discard the flow-through and reuse the collection tube.
Two loading rounds are expected because the binding mixture volume is larger than a single column load.
Add 500 µl Buffer DW1 to the column and centrifuge at 12,000 × g for approximately 1 min. Discard the flow-through.
This wash removes proteins and plant-derived contaminants remaining after binding.
Add 500 µl Buffer GW2 to the column and centrifuge at 12,000 × g for approximately 1 min. Discard the flow-through.
Ensure ethanol has been added to Buffer GW2 before use. An optional repeat GW2 wash can be used when purity is more important than the shortest processing time.
Centrifuge the empty column at 12,000 × g for 2 min to dry the membrane.
Residual ethanol may inhibit PCR or enzymatic downstream reactions. After drying, avoid leaving the membrane excessively dry before elution.
Place the column into a clean 1.5 ml tube. Add 40–75 µl Buffer AE preheated to 65°C directly to the center of the membrane, let stand for 2 min, and centrifuge at 12,000 × g for 1 min.
Preheated AE and center-membrane loading improve recovery from plant genomic DNA bound to the membrane.
Add another 40–75 µl preheated Buffer AE to the membrane, let stand for 2 min, centrifuge for 1 min, discard the column and store DNA at 2–8°C or at −20°C for long-term storage.
The second elution is shown because it is listed as the subsequent elution step in the manual. It improves total recovery, while a smaller combined volume may be selected when concentration is the priority.
Use the Protocol B PAL / PVP-40 route for polyphenol-rich plant or fungal samples. Grind the sample under liquid nitrogen and transfer 50–100 mg fresh / frozen sample or 15–30 mg dry sample into a 2 ml tube.
Before use, prepare the PAL / PVP route as specified in the manual: add PVP-40 to Buffer PAL at 2% (w/v), mix thoroughly, and add 2-mercaptoethanol at 1% (v/v) before use. The PAL / PVP mixture should not be kept at room temperature for more than 1 week. Keep sample input conservative for the first run.
Add 700 µl preheated Buffer PAL containing 2% (w/v) PVP-40 and 1% (v/v) 2-mercaptoethanol, vortex fiercely to disperse the sample thoroughly, and incubate at 65°C. Mix by inversion during incubation.
The manual allows 15–30 min. The displayed standard timeline uses the shortest reasonable path; difficult or highly oxidizing samples may require the longer end of the range.
Add 700 µl chloroform, vortex for 15 sec, and centrifuge at 12,000 × g for 5 min at room temperature.
For strongly polyphenol- or starch-rich matrices, the manual allows an additional phenol:chloroform extraction before this step. If selected, treat it as an add-on clarification step; it is not included in the standard left-side timeline.
Carefully transfer 600 µl cleared supernatant to a new centrifuge tube without disturbing the interphase.
Clean phase recovery is the main purity control point in this workflow. Optional RNase A treatment, when required, adds about 15 min.
Add 300 µl Buffer GWP and 600 µl 100% ethanol to the supernatant. Invert 10–15 times to mix thoroughly; if a sediment pellet appears, disperse it by pipetting up and down.
Unlike Protocol A, this route uses ethanol together with GWP to establish the column-binding condition after polyphenol-control lysis.
Insert a HiPure gDNA Mini Column II into a 2 ml collection tube. Load up to 750 µl mixture and centrifuge for 1 min. Discard the flow-through.
Keep any precipitate suspended while loading so that DNA-containing material is not left behind in the tube.
Load the remaining mixture onto the column and centrifuge for 1 min. Discard the flow-through and reuse the collection tube.
Repeat loading is expected because the binding mixture exceeds one column load.
Add 500 µl Buffer DW1 and centrifuge at 12,000 × g for approximately 1 min. Discard the flow-through.
This wash helps remove residual lysis and plant-matrix contaminants.
Add 500 µl Buffer GW2 and centrifuge at 12,000 × g for approximately 1 min. Discard the flow-through.
For colored membranes or demanding downstream use, an additional GW2 or ethanol wash may be selected according to the manual troubleshooting guidance.
Centrifuge the empty column at 12,000 × g for 2 min to dry the membrane.
Residual ethanol is a common cause of poor enzymatic performance in downstream reactions.
Transfer the column to a clean 1.5 ml tube. Add 40–75 µl Buffer AE preheated to 65°C to the membrane center, let stand for 2 min, and centrifuge for 1 min.
Use a volume compatible with both yield and downstream concentration requirements.
Repeat elution with another 40–75 µl preheated Buffer AE, centrifuge for 1 min, discard the column and store DNA at 2–8°C or −20°C for long-term storage.
A second elution improves total recovery, especially when plant genomic DNA remains strongly retained on the membrane.
This workflow separates sample-specific liquid-nitrogen disruption and CTAB / PAL lysis from the shared chloroform clarification and silica-column purification route. It is intended as a practical companion to the product manual rather than a replacement for the official protocol. Protocol A is used for general plant or fungal samples, while Protocol B adds a PVP-assisted lysis route for polyphenol-rich material. The downstream workflow follows organic extraction, cleared DNA-containing supernatant recovery, binding-condition adjustment, column loading, DW1 / GW2 washing, membrane drying and two-step AE elution.
| Sample route | Displayed preparation estimate | Typical manual workflow time |
|---|---|---|
| Protocol A · Standard Plant / Fungus | 5 min disruption + 20 min PAL lysis | 50–60 min |
| Protocol B · Polyphenol-Rich Samples | 6 min disruption + 15–30 min PAL / PVP lysis | 50–75 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, pipetting, tube transfer, centrifuge handling, cleared-supernatant recovery, column loading, filtrate disposal, column repositioning, membrane drying, elution and final tube transfer. For short protocol ranges, the timeline uses the midpoint or a near-midpoint value. For long or optional protocol ranges, the displayed standard timeline uses the shortest reasonable path, while the note and total-time range indicate where extended handling may apply. For this D3187 workflow, Protocol A has a relatively fixed timing structure, with variation mainly from grinding quality, powder dispersion, phase-boundary handling and column transfer. Protocol B retains a wider total-time range because its PAL / PVP lysis step is specified as 15–30 min; optional RNase A treatment, phenol:chloroform extraction or additional washing should be treated as extra handling outside the displayed standard timeline.
D3187 uses CTAB / PAL lysis followed by chloroform clarification to separate plant debris, proteins, polysaccharides and other matrix components before genomic DNA is bound to a silica membrane. Protocol A allows 2-mercaptoethanol adjustment for difficult samples, while Protocol B uses 2% (w/v) PVP-40 and 1% (v/v) 2-mercaptoethanol in PAL to control polyphenol-related oxidation and inhibitor carryover.
The most important control points are sample amount, complete powdering under liquid nitrogen, correct PAL additive concentration, rapid dispersion in preheated PAL, clean recovery of the DNA-containing cleared supernatant and adequate ethanol removal before elution. Avoid overloading the column, do not carry chloroform-rich material into the binding step, disperse precipitate before loading, confirm ethanol addition to GW2, and apply preheated AE directly to the membrane center for consistent recovery.