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Novel Plant gDNA Extraction Maxi 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 1 g fresh or 200 mg dry plant samples within approximately 1 hour. 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, cells, fungi, and other samples.

Operation Steps:
Tips:
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 (maximum processing capacity: no more than 1 g of fresh tissue or 200 mg of dry tissue) and grind it thoroughly into fine powder in a mortar with liquid nitrogen.

2.Transfer the fine powder to a 15 mL centrifuge tube (do not thaw). Add 5 mL of preheated Buffer AP1 (65℃) and 40 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 10-15 minutes, inverting the centrifuge tube 2-3 times during incubation to mix the sample.

4.Add 1.8 mL of Buffer AP2, mix thoroughly, incubate on ice for 10 minutes, then centrifuge at 9,000 x g at room temperature for 10 minutes. Carefully aspirate the supernatant into a new 50 mL centrifuge tube, taking care not to aspirate the interface material.
If a high-speed centrifuge is not available, centrifugation at 4,000-5,000 x g is acceptable with an appropriate extension of centrifugation time.

5.Calculate the volume of the supernatant, add 1.5 volumes of Buffer AP3/E (ensure anhydrous ethanol has been added!), and immediately vortex to mix thoroughly. 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 vortex 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 3,000-5,000 x g for 5 minutes and discard the waste liquid in the collection tube.

7.Add 10 mL of Wash Buffer WB (ensure anhydrous ethanol has been added!), centrifuge at 3,000-5,000 x g for 4 minutes, and discard the waste liquid.

8.Add 10 mL of Wash Buffer WB (ensure anhydrous ethanol has been added!), centrifuge at 3,000-5,000 x g for 3 minutes, and discard the waste liquid.

9.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 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.

10.Remove the Spin AC column and place it into a clean centrifuge tube. Add 1-2 mL 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 5 minutes, then centrifuge at 3,000-5,000 x g for 3 minutes to obtain DNA. Additionally, transfer the eluted solution back into the spin column, incubate at room temperature for 2 minutes, and elute again to improve concentration and yield.
Larger elution volumes result in higher elution efficiency. To increase yield, increase the elution volume. For higher DNA concentration, reduce the elution volume (minimum 500 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.