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Novel Plant gDNA Extraction Kit(SpinColumn)

Packing Specification：

Product Introduction:
This kit adopts DNA adsorption columns and a novel, unique buffer system, suitable for the rapid and simple extraction of genomic DNA from plant samples containing phenols, polysaccharides, and enzyme inhibitors. It can purify DNA from one or more 100 mg fresh or 20 mg dry plant samples within 30 minutes. The extraction process does not require toxic organic solvent extraction such as phenol-chloroform, nor time-consuming isopropanol or ethanol precipitation. It can quickly and efficiently remove impurities such as polysaccharides, phenols, and enzyme inhibitors. The purified DNA can be directly used for PCR, enzyme digestion, hybridization, and other experiments.
After grinding, fresh or dry plant tissues (cells) are lysed with lysis buffer; proteins, polysaccharides, and cell debris are removed by precipitation. 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, phenols, 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, 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 plant tissues, leaves, roots, stems, whole plants, plant tissue cells, fungi, and other samples.

Operation Steps:
Note: Before first use, add the specified amount of anhydrous ethanol to Wash Buffer WB and Buffer AP3/E, mix thoroughly, and mark the box to indicate ethanol has been added to avoid repeated addition!
1.Take an appropriate amount of plant tissue (100 mg of fresh tissue or 20 mg of dry tissue; a slightly larger amount can be taken to compensate for losses adhering to the mortar) and grind it thoroughly into fine powder in a mortar with liquid nitrogen. Before grinding, prepare a 1.5 mL centrifuge tube, add 400 μL of Buffer AP1 and 4 μL of RNase A (10 mg/mL), and set aside at room temperature.

2.Transfer the fine powder (100 mg of fresh tissue or 20 mg of dry tissue) to the pre-prepared 1.5 mL centrifuge tube (containing 400 μL of Buffer AP1 and 4 μL of RNase A (10 mg/mL)), vortex to mix thoroughly to assist lysis.
If tissue lysis is difficult, a gentle homogenization step for 10 seconds can be added as needed to aid lysis. In most cases, centrifugation to remove incompletely lysed tissue is not required, as there is a subsequent centrifugation step for impurity removal.

3.Incubate in a 65℃ water bath for 10 minutes, inverting the centrifuge tube 2-3 times during incubation to mix the sample.

4.Add 130 μL of Buffer AP2, mix thoroughly, incubate on ice for 5 minutes, then centrifuge at 14,000 rpm for 5-10 minutes. Carefully aspirate the supernatant into a new 1.5 mL centrifuge tube, taking care not to aspirate the interface material.

5.Calculate the volume of the supernatant, add 1.5 volumes of Buffer AP3/E (ensure anhydrous ethanol has been added!), and immediately pipette to mix. Flocculent precipitation may occur after adding Buffer AP3/E, but it will not affect DNA extraction. Note: Add Buffer AP3/E directly to the supernatant and pipette to mix immediately.

6.Load the mixture from the previous step (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. (First load 650 μL, centrifuge, discard the waste liquid, then add the remaining solution and centrifuge again.)

7.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.

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

9.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).

10.Remove the Spin AC column and place it into a clean centrifuge tube. Add 100 μL of Elution Buffer EB (the elution buffer can be preheated in a 65-70℃ water bath beforehand) 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. To increase yield, increase the elution volume. For higher DNA concentration, reduce the elution volume (minimum 50 μL; volumes smaller than this reduce elution efficiency and DNA yield).

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