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CTAB Plant gDNA Extraction Maxi Kit(SpinColumn)

Packing Specification：

Product Introduction:
The improved classic CTAB plant DNA extraction buffer (supplemented with multiple components targeting plant-specific polysaccharide and polyphenol removal) rapidly lyses cells and inactivates intracellular nucleases. After chloroform extraction, polysaccharides, polyphenols, and proteins are removed by centrifugation. 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, polysaccharides, polyphenols, cellular metabolites, proteins, and other impurities are further removed. 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 1 hour.
4.Multiple polysaccharide and polyphenol removal components and repeated 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 large quantities of plant genomic DNA.

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!
Preheat the required amount of Lysis Buffer PL at 65℃. Add β-mercaptoethanol to a final concentration of 2% before use.

1.Take an appropriate amount of plant tissue (1-2 grams of fresh tissue or 0.3-0.4 grams of dry tissue) and grind it thoroughly into fine powder in a mortar with liquid nitrogen.

2.Transfer the fine powder to a 50 mL centrifuge tube (do not thaw). Add 10 mL of preheated Lysis Buffer PL (65℃) (confirm β-mercaptoethanol has been added to 2%), vortex vigorously to mix, and gently pipette with a large-bore pipette tip to assist lysis.
If tissue lysis is difficult, a gentle homogenization step for 10 seconds can be added as needed to aid lysis.

3.Incubate in a 65℃ water bath for 30-60 minutes, inverting the centrifuge tube several times during incubation to mix the sample.
Optional: If the tissue is dry or the yield is low, the water bath time can be appropriately extended. If RNA residue is high, add 100 μL of RNase (20 mg/mL) before water bath incubation.Note: If residual RNA in the extracted DNA causes abnormal electrophoresis phenomena such as band smearing, distortion, or high background, add 1% RNase (10 mg/mL) and incubate at 37℃ or room temperature for 30 minutes to digest the RNA. No special treatment is required after digestion, and the DNA can be used directly for PCR or enzyme digestion.

4.Add 10 mL of chloroform or chloroform/isoamyl alcohol (mixed at a volume ratio of 24:1), invert to mix thoroughly for a few minutes (or vortex to mix), then centrifuge at ≥ 9,000 x g for 10 minutes.
For plant tissues rich in polysaccharides and polyphenols, extract once with an equal volume of phenol/chloroform (1:1) before Step 4.

5.Carefully aspirate the supernatant into a new 50 mL centrifuge tube, taking care not to aspirate the interface material.
If the supernatant is turbid, repeat Step 4 until a clear supernatant is obtained.

6.Estimate the volume of the supernatant accurately. Add 1.5 volumes of Binding Buffer PQ (ensure anhydrous ethanol has been added!) and immediately vortex to mix thoroughly. Precipitation may occur at this step, but it will not affect the experimental results.

7.Load the mixture from the previous step (including any precipitation) into a Spin AC column (placed in a collection tube). Let it stand for 2 minutes, centrifuge at 9,000 x g for 2 minutes, and discard the waste liquid in the collection tube. (A maximum of 20 mL of the mixture can be loaded per centrifugation; discard the waste liquid, add the remaining solution, and centrifuge again.)

8.Add 10 mL of Inhibitor Removal Buffer IR, centrifuge at 9,000 x g for 2 minutes, and discard the waste liquid.

9.Add 10 mL of Wash Buffer WB (ensure anhydrous ethanol has been added!), centrifuge at 9,000 x g for 2 minutes, and discard the waste liquid.

10.Repeat Step 9 once.

11.Place the Spin AC column back into the empty collection tube. Centrifuge at maximum speed (preferably ≥ 9,000 x g; if the centrifuge speed is low, extend the centrifugation time accordingly) for 10-15 minutes to dry residual ethanol on the membrane matrix. Use a pipette tip to remove any residual ethanol between the inner ring pressure ring and the column wall, then air-dry at room temperature or in an oven for a few minutes.
Note: This step is intended to completely remove residual ethanol in the spin column. Residual ethanol inhibits downstream reactions and significantly reduces elution efficiency and DNA yield. If the elution yield is low, Step 12 must be performed.

12.Remove the Spin AC column and place it into a clean centrifuge tube. Add 1.5-2 mL 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 9,000 x g for 4-5 minutes. If a larger amount of DNA is needed, transfer the eluted solution back into the spin column and centrifuge for 2 minutes.
Larger elution volumes result in higher elution efficiency. To increase yield, increase the elution volume. For higher DNA concentration, reduce the elution volume (minimum 1 mL; volumes smaller than this reduce elution efficiency and DNA yield).

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