Collect yeast pellet
Transfer 1–1.5 ml yeast culture to a 2.0 ml centrifuge tube. Centrifuge at 5,000 × g for 5 min to collect yeast cells and remove the supernatant.
The manual specifies yeast input below 5 × 107 cells for this kit.
HiPure Yeast DNA Kit — Yeast Cell Wall Lysis Workflow Note
Cat. No. D314701 / D314702 / D314703
Standard yeast DNA extraction route with enzymatic cell-wall digestion, optional mechanical reinforcement and silica column purification.
Yeast collection & cell-wall pretreatment
Optional reinforcement
Shared silica column purification
6 min
Cumulative 6 min
31 min
Cumulative 37 min
SE / 2-mercaptoethanol / lyticase treatment
Add 300 μl Buffer SE, 10 μl 2-mercaptoethanol and 30 μl Lyticase to the pellet. Fully resuspend by vortexing and incubate with shaking at 37°C for 30–60 min.
The displayed timeline uses the shortest reasonable path for this long manual range: 30 min incubation plus routine resuspension and tube handling. Extending lyticase treatment to 60 min is reflected in the upper end of the total-time range.
5 min
Cumulative 42 min
Recover treated yeast pellet
Centrifuge at 5,000 × g for 5 min and remove the supernatant.
2 min
Cumulative 44 min
ATL resuspension
Add 300 μl Buffer ATL to the pellet and fully resuspend by vortexing.
If Buffer ATL has precipitated at low temperature, redissolve it before use.
+15 min
Optional
Optional RNA control
If RNA-free genomic DNA is required, add 10 μl RNase A to the sample and incubate for 15 min at room temperature.
RNase A is not provided with the kit and this step is not included in the standard cumulative timeline.
+5 min
Optional
Optional glass-bead reinforcement
For further lysis of yeast cells, add approximately 300 mg glass beads and 2 μl Reagent DX, then vortex at maximum speed for 5 min.
This step is useful when enzymatic pretreatment alone may not provide sufficient disruption; it is not included in the standard cumulative timeline.
11 min
Cumulative 55 min
DL / Proteinase K digestion
Add 300 μl Buffer DL and 10 μl Proteinase K to the sample. Fully vortex and incubate in a 70°C water bath for 10 min.
Add Proteinase K directly to the sample during this step; do not add it directly to Buffer DL for storage or premixing.
4 min
Cumulative 59 min
Clarify lysate and transfer supernatant
Centrifuge at 10,000 × g for 3 min to remove undigested impurities. Transfer 500 μl supernatant to a new centrifuge tube.
1 min
Cumulative 60 min
Ethanol binding adjustment
Add 250 μl absolute ethanol to the supernatant and vortex for 10 s.
Ethanol must be added before loading the lysate onto the column.
2 min
Cumulative 62 min
Column binding
Insert a HiPure DNA Mini Column I into a 2 ml Collection Tube. Transfer the mixture to the column and centrifuge at 10,000 × g for 30–60 s.
The timeline includes routine loading and collection-tube handling by an experienced operator.
2 min
Cumulative 64 min
GW1 wash
Discard the flow-through, reuse the collection tube and add 500 μl Buffer GW1 diluted with absolute ethanol. Centrifuge at 10,000 × g for 30–60 s.
2 min
Cumulative 66 min
GW2 wash
Add 600 μl Buffer GW2 diluted with absolute ethanol and centrifuge at 10,000 × g for 30–60 s.
Confirm that ethanol has been added to Buffer GW2 before use.
3 min
Cumulative 69 min
Dry spin
Discard the flow-through, reuse the collection tube and centrifuge at 13,000 × g for 2 min to reduce Buffer GW2 carryover.
Residual ethanol can interfere with downstream enzymatic reactions.
5 min
Cumulative 74 min
Elution
Place the column in a clean 1.5 ml centrifuge tube. Add 30–50 μl Buffer AE preheated to 65°C to the center of the membrane. Incubate at room temperature for 3 min and centrifuge at 10,000 × g for 1 min.
A longer 5 min membrane incubation or a second elution with 100–200 μl Buffer AE can increase yield but is not included in the standard timeline.
1 min
Cumulative 75 min
Store purified DNA
Discard the column and store DNA at 2–8°C for short-term storage. For long-term storage, keep DNA at −20°C or −80°C.
Typical processing time, standard route
75–105 min
1. Workflow structure
This workflow separates yeast collection and cell-wall pretreatment from the shared silica column purification path. It is intended as a practical companion to the product manual rather than a replacement for the official protocol. The workflow focuses on the standard protocol for yeast DNA extraction; optional RNase treatment and glass-bead reinforcement are shown as conditional steps.
2. Time interpretation
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, pellet resuspension, supernatant removal, residual-liquid removal, column repositioning, drying control, 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 D3147, the manual 30–60 min lyticase treatment is therefore shown using the 30 min path plus routine handling; the longer 60 min incubation is reflected in the upper end of the total-time range rather than in the displayed cumulative line.
3. Workflow characteristics
D3147 uses a yeast-specific cell-wall pretreatment before proteinase digestion and silica membrane purification. The lyticase step at 37°C weakens the yeast cell wall before ATL resuspension and DL / Proteinase K digestion. For difficult yeast cells, glass beads and Reagent DX can be added as a mechanical reinforcement step, but this is treated as optional rather than part of the standard cumulative timeline.
4. Practical considerations
The key handling point is complete resuspension of the yeast pellet before the lyticase step and again before ATL / DL lysis. Proteinase K should be added directly to the sample during the DL lysis step; do not pre-mix or store Proteinase K directly in Buffer DL, as this may reduce protease activity. Buffer ATL may precipitate at low temperature and should be redissolved before use if precipitation is observed.