Products

Tissue/Cell gDNA Extraction Kit(SpinColumn)

Packing Specification：

Product Introduction:
The unique combination of Binding Buffer and Proteinase K rapidly lyses 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 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, and the DNA length can reach 30 kb - 50 kb, 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 animal and plant tissues and cells.

Operation Steps:
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!
Pretreat the spin column with Equilibration Buffer (mandatory step; see "Use of Equilibration Buffer" above for detailed method).

1.Cultured Tissue Cells
a.Collect approximately 105-106 suspension cells into a 1.5 mL centrifuge tube. For adherent cells, digest with trypsin first, then pipette to detach and collect.
b.Centrifuge at 13,000 rpm for 10 seconds to pellet the cells. Discard the supernatant, leaving the cell pellet with approximately 10-20 μL of residual liquid.
c.Add 200 μL of 1×PBS to resuspend and wash the cells. Centrifuge at 13,000 rpm for 10 seconds to pellet the cells. Aspirate and discard the supernatant completely, then resuspend the cell pellet in 180 μL of 1×PBS.
d.Add 20 μL of Proteinase K (20 mg/mL) solution, mix thoroughly. Add 200 μL of Binding Buffer CB, immediately vortex to mix well, and incubate at 70℃ for 10 minutes.
Optional step: If significant RNA contamination is present, add 20 μL of RNase A (25 mg/mL) solution before adding 200 μL of Binding Buffer CB, vortex to mix, and incubate at room temperature for 5-10 minutes.
e.After cooling, add 100 μL of isopropanol, immediately vortex to mix thoroughly (flocculent precipitation may occur).
f.Load the mixture (including any precipitation) into a Spin AC column (placed in a collection tube). Centrifuge at 13,000 rpm for 60 seconds and discard the waste liquid in the collection tube.
g.Proceed to Step 4 under "Common Operation Steps".

2.Animal and Plant Tissues (e.g., Mouse Liver, Brain, or Plant Leaves)
a.Grind fresh or thawed tissue into fine powder in liquid nitrogen, or cut into small pieces with a scalpel (cutting into small pieces can improve yield). Take 20-50 mg and transfer into a 1.5 mL centrifuge tube containing 180 μL of Tissue Lysis Buffer TL. Pipette to mix with a large-bore pipette tip.
b.Add 20 μL of Proteinase K (20 mg/mL) solution, immediately vortex to mix thoroughly.
c.Incubate the lysate in a 55℃ water bath for 1-3 hours or until complete tissue digestion, gently shaking several times during incubation to assist lysis.
Optional step: If significant RNA contamination is present, add 20 μL of RNase A (25 mg/mL) solution after completing Step 3, vortex to mix, and incubate at room temperature for 5-10 minutes.
d.Add 200 μL of Binding Buffer CB, immediately vortex to mix thoroughly, and incubate at 70℃ for 10 minutes.
e.After cooling, add 100 μL of isopropanol, immediately vortex to mix thoroughly (flocculent precipitation may occur).
f.Use a 1 mL pipette tip to aspirate the mixture and load it into a Spin AC column (placed in a collection tube). Centrifuge at 13,000 rpm for 60 seconds and discard the waste liquid in the collection tube.
If insoluble tissue debris clogs the pipette tip, gently rub the tip on absorbent paper to remove the debris. If only a small amount of mixture is aspirated, discard the tip and debris together to avoid clogging the spin column.
g.Proceed to Step 4 under "Common Operation Steps".

3.Animal Tissue (Mouse Tail)
a.Cut 0.2-0.5 cm of the mouse tail tip (i.e., 20-50 mg; ensure to cut within 0-2 cm of the tail tip for optimal lysis), or grind the tissue into fine powder in liquid nitrogen. Transfer into a 1.5 mL centrifuge tube containing 180 μL of Tissue Lysis Buffer TL. Pipette to mix with a large-bore pipette tip.
b.Add 20 μL of Proteinase K (20 mg/mL) solution, immediately vortex to mix thoroughly.
c.Incubate the lysate in a 55℃ water bath for 3 hours or until complete tissue digestion, gently shaking several times during incubation to assist lysis.
Optional step: If significant RNA contamination is present, add 20 μL of RNase A (25 mg/mL) solution after completing Step 3, vortex to mix, and incubate at room temperature for 5-10 minutes.
d.Aspirate the lysate 2-3 times with a 1 mL disposable syringe without a needle.
e.Add 200 μL of Binding Buffer CB and 100 μL of isopropanol, immediately vortex to mix thoroughly.
f.Centrifuge at 13,000 rpm for 5 minutes. Load the supernatant 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.
g.Proceed to Step 4 under "Common Operation Steps".
Note: Immediate vortexing or pipetting to mix thoroughly is critical in the above steps. Inadequate mixing will severely reduce yield. For viscous samples that are difficult to mix, vortex for 15 seconds if necessary.

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

5.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.

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

7.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).

8.Remove the Spin AC column and place it into a clean centrifuge tube. Add 100 μL of Elution Buffer TE (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).

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