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Yeast gDNA Extraction Kit(SpinColumn,Lytic Enzyme,Protein K)

Packing Specification：

Product Introduction:
This kit adopts DNA adsorption columns and a unique buffer system, suitable for the rapid and simple extraction of genomic DNA from yeast cultures of various sources. Typically, 10-15 μg of high-quality genomic DNA can be purified from approximately 3 mL of yeast culture in the exponential growth phase in one extraction. The purified DNA can be directly used for PCR, enzyme digestion, hybridization, and other experiments. After yeast cells are treated with Lytic Enzyme to remove the cell wall, the unique combination of Binding Buffer and Proteinase K rapidly lyses the cells and inactivates intracellular nucleases. Genomic DNA is then selectively adsorbed onto the silica membrane of the spin column under high chaotropic salt conditions. Through a series of rapid washing and centrifugation steps, Inhibitor Removal Buffer and Wash Buffer remove impurities such as cellular metabolites and proteins. Finally, pure genomic DNA is eluted from the silica membrane using a low-salt Elution Buffer.

Product Features:
1.The silica membrane in the spin column is made of high-quality special adsorption membrane, ensuring minimal variation in adsorption capacity between columns.
2.No need for toxic reagents such as phenol or ethanol precipitation steps.
3.Fast and simple; operation after cell lysis for a single sample can generally be completed within 30 minutes.
4.Multiple column washes ensure high purity. The typical OD260/OD280 ratio is 1.7-1.9, which can be directly used for PCR, Southern blot, and various restriction enzyme digestion reactions.

Application Scope:
Suitable for the rapid extraction of genomic DNA from various yeasts.

Operation Steps:
Tips:
Before first use, add the specified amount of anhydrous ethanol to Wash Buffer WB, mix thoroughly, and mark the box to indicate ethanol has been added to avoid repeated addition!
Add 0.2% β-mercaptoethanol to the required volume of Sorbitol buffer and bring to room temperature for later use.

1.Take 1-3 mL of yeast culture (no more than 3×10⁷ cells, preferably in the early exponential growth phase), centrifuge at 12,000 rpm for 30 seconds. Aspirate and discard the supernatant as much as possible to collect the yeast pellet.
For more than 1.5 mL of culture, centrifuge to discard the supernatant, add more culture to the same 1.5 mL tube, and repeat Step 1 until sufficient yeast pellet is collected.

2.Add 300 μL of Sorbitol buffer and gently pipette to fully resuspend the cells. Add 50 μL of Lytic Enzyme stock solution, invert thoroughly to mix, and incubate at 37℃ for 1-3 hours to digest the cell wall (invert several times during incubation to assist digestion).
Alternative lysis methods for yeasts incompatible with Lytic Enzyme: If cell wall digestion is incomplete leading to low yield, increase the dosage of Lytic Enzyme to improve the working concentration, extend the digestion time, or raise the temperature to 45℃. For yeasts not suitable for Lytic Enzyme digestion, other methods such as vortexing with 0.5 mm glass beads or repeated freeze-thaw cycles can be used. Glass bead method: Add 180 μL of Buffer YB to the yeast pellet to thoroughly resuspend, add 0.1 g of acid-washed glass beads (0.45-0.55 mm in diameter), vortex vigorously for 10 minutes, let stand for a few minutes to allow the glass beads to settle, carefully aspirate the supernatant into a new tube, and proceed to Step 4.

3.Centrifuge at 13,000 rpm for 1 minute. Aspirate and discard the supernatant as much as possible, add 180 μL of Buffer YB to fully resuspend the cell pellet.

4.Add 20 μL of Proteinase K solution (20 mg/mL) and immediately vortex to mix thoroughly.

5.Incubate the mixture in a 55℃ water bath until complete digestion, gently shaking several times during incubation to assist lysis.
The required digestion time depends on the yeast quantity, strain, and growth status, generally 15 minutes. Overnight digestion is also acceptable if convenient.
Optional step (generally not required): If significant RNA contamination is present, add 20 μL of RNase A (25 mg/mL) solution after completing Step 5, vortex to mix, and incubate at room temperature for 5-10 minutes.

6.Add 200 μL of Binding Buffer CB, immediately vortex to mix thoroughly, and incubate at 70℃ for 10 minutes.

7.After cooling, add 100 μL of isopropanol and immediately vortex to mix thoroughly (flocculent precipitation may occur).
8.Load the mixture (including any precipitation) into a Spin AC column (placed in a collection tube). Centrifuge at 13,000 rpm for 30-60 seconds and discard the waste liquid in the collection tube.

9.Add 500 μL of Inhibitor Removal Buffer IR, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid.

10.Add 600 μL of Wash Buffer WB (ensure anhydrous ethanol has been added!), centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid.

11.Add 600 μL of Wash Buffer WB, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid.

12.Place the Spin AC column back into the empty collection tube and centrifuge at 13,000 rpm for 2 minutes to thoroughly remove residual Wash Buffer (residual ethanol may inhibit downstream reactions).

13.Remove the Spin AC column and place it into a clean centrifuge tube. Add 100 μL of Elution Buffer EB (preheating the elution buffer in a 65-70℃ water bath beforehand improves elution efficiency) to the center of the adsorption membrane. Incubate at room temperature for 3-5 minutes, then centrifuge at 12,000 rpm for 1 minute. Transfer the eluted solution back into the spin column, incubate at room temperature for 2 minutes, and centrifuge at 12,000 rpm for 1 minute.
Larger elution volumes result in higher elution efficiency. For higher DNA concentration, reduce the elution volume (minimum 50 μL; volumes smaller than this reduce elution efficiency and DNA yield).

14.Store DNA at 2-8℃ for short-term use or -20℃ for long-term storage.