SDS lysis, salt precipitation and silica-column purification for plant and fungal DNA
Cat.No. D316402 / D316403 · Manual workflow overview for plant and fungal tissue
Disrupt plant or fungal tissue by liquid-nitrogen grinding or another bead-beat method. Transfer up to 100 mg wet tissue or 20 mg dried tissue into the tube, keeping the powder from thawing before lysis.
Sample excess and incomplete disruption are the main causes of viscous lysate and membrane clogging.
Add 600 µl Buffer SPL and 10 µl RNase A. Vortex vigorously and pipet if needed until clumps are dispersed.
Do not premix Buffer SPL and RNase A before use.
Incubate at 65°C for 10 minutes. Invert the tube 2–3 times during incubation to maintain contact between the sample powder and lysis buffer.
RNase A acts during lysis so RNA is removed before column binding.
Add 200 µl Buffer PS, vortex to mix, and incubate on ice for 5 minutes.
This step precipitates proteins, polysaccharides and cell-debris components before clarification.
Centrifuge for 5 minutes at >13,000 × g to pellet cell debris and precipitated contaminants.
A firm pellet and clear supernatant are important for stable column flow.
Transfer 600 µl of the supernatant to a new tube without disturbing the cell-debris pellet.
Do not carry over the pellet; it can reduce purity and clog the membrane.
Add 1.5 volumes of Buffer GW1 to the cleared lysate, for example 900 µl Buffer GW1 to 600 µl supernatant, and mix immediately by pipetting.
A precipitate may form after GW1 addition; include it during column loading as described in the protocol.
Insert the HiPure gDNA Mini Column into a 2 ml collection tube. Load 750 µl of the mixture and centrifuge for 1 minute at ≥10,000 × g. Discard the flow-through.
Make sure ethanol has been added to Buffer GW1 before use.
Load the remaining mixture onto the same column and centrifuge again. Discard the flow-through and reuse the collection tube.
Load in two portions rather than overfilling the column.
Add 500 µl Buffer GW1 and centrifuge for 1 minute at ≥10,000 × g. Discard the flow-through.
This wash continues removal of salts and soluble contaminants under binding-compatible conditions.
Add 650 µl Buffer GW2 and centrifuge for 1 minute at ≥10,000 × g. Discard the flow-through.
Ensure ethanol has been added to Buffer GW2 before use.
Add another 650 µl Buffer GW2 and centrifuge for 1 minute at 10,000 × g. Discard the flow-through.
If the membrane remains strongly colored, an additional 96–100% ethanol wash can be used as an optional cleanup step.
Centrifuge the empty column for 1 minute at 10,000 × g to remove residual Buffer GW2.
Residual ethanol or wash buffer may inhibit downstream enzymatic reactions.
Transfer the column to a clean microcentrifuge tube. Add 50–100 µl Buffer AE directly to the membrane, incubate for 5 minutes, and centrifuge for 1 minute to elute DNA.
For larger DNA loads, 200 µl AE can improve total recovery but gives a more diluted eluate.
Repeat the elution once. Store the purified DNA at -20°C.
Use a new tube to keep the second eluate separate, or reuse the first tube to combine eluates.
This workflow follows the manual plant and fungal tissue route for HiPure SF Plant DNA Kit. It separates the front-end sample disruption and SPL lysis stage from the PS salt-precipitation clarification step and the shared silica-column binding, wash and elution route. It is intended as a practical companion to the product manual rather than a replacement for the official protocol.
Protocol times stated in the product manual are retained where applicable. Steps without explicit timing are estimated for an experienced operator, including pipetting, tube transfer, centrifuge handling, 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. Optional cleanup steps are described in the notes but are not included in the standard total time. For this D3164 workflow, the protocol timing is relatively fixed, and the 55–65 min total mainly reflects cumulative manual differences in sample disruption, SPL dispersion, clean supernatant recovery, immediate GW1 mixing, column handling and residual wash-buffer removal.
D3164 is a non-organic plant DNA workflow. Buffer SPL lyses the disrupted plant or fungal material while RNase A digests RNA during the lysis stage. Buffer PS then precipitates proteins, polysaccharides and cell-debris components, allowing the DNA-containing cleared supernatant to be adjusted with Buffer GW1 for silica-column binding. The downstream route uses one GW1 wash, two GW2 washes, a dry spin and two AE elutions.
Do not exceed the recommended input amount, and keep the sample powder from thawing before SPL lysis. Buffer SPL and RNase A should not be premixed before use. After PS treatment and centrifugation, transfer the cleared supernatant without disturbing the cell-debris pellet. Buffer GW1 should be added directly to the supernatant and mixed immediately; any precipitate formed after GW1 addition should be loaded according to the protocol. Ethanol must be added to GW1 and GW2 before use, and the final dry spin should be sufficient to reduce GW2 carryover before AE elution.