Sample4 rxns ($14.4)
Sample4 rxns ($14.4)
Note: PureDireX / Purification Kits / Extraction Kit / Plant
The Plant Plant Genomic DNA Isolation Reagent provides an easy 3-step method to isolate a high yield of total DNA (including genomic, mitochondrial, and chloroplast DNA from the plant tissue and cells). This unique reagent is able to lyse the most common plant samples as well as the samples high in polysaccharides. If a large sample is required, the reagent volume can be scaled proportionately, making this reagent not only very user-friendly but also highly versatile. The DNA phenol extraction is not required, and the entire procedure can be completed in 90 minutes. The extracted total DNA is ready for use in PCR, Real-time PCR, Southern Blotting, Mapping, and RFLP.
The standard protocol uses the PG Reagent for lysis of plant samples. For most common plant species, the reagent system ensures the isolated plant genomic DNA with a high yield and good quality.
1. Cut off 100 mg of the fresh plant tissue or 50 mg of the dry plant tissue.
2. Grind the sample under liquid nitrogen to a fine powder using a mortar and pestle.
1. Add 1 ml of the PG Reagent and 0.5 μl of the RNase A (50 mg/ml) to the sample in the mortar and grind the sample until it is completely dissolved.
2. Transfer the dissolved sample to a 1.5 ml microcentrifuge tube.
3. Incubate at 65°C for 30-50 minutes. Centrifuge at 14-16,000 x g for 10 minutes. Transfer he supernatant to a new 1.5 ml microcentrifuge tube.
Add 600 μl of the chloroform to the supernatant from Step 1.
Add a 1/10 volume of the PG Reagent and 600 μl of the chloroform to the supernatant from Step 1.
1. Shake vigorously and then centrifuge at 14-16,000 x g for 10 minutes.
2. Carefully remove the upper phase and transfer it to a new 1.5 ml microcentri fuge tube.
3. Repeat the Phase Separation Step until the interphase becomes clear, then transfer the clear upper phase to a new 1.5 ml microcentrifuge tube.
NOTE: The number of repetitions is dependent on the sample type; e.g. dense tissue samples may require a higher number of repeats.
1. Add 800 μl of the isopropanol to the 1.5 ml microcentrifuge tube containing the clear upper phase from step 2.
2. Mix the sample by inverting gently and letting it stand for 5 minutes at the room temperature (DNA precipitation can be increased with the extended standing time).
3. Centrifuge at 14-16,000 x g for 20 minutes.
4. Discard the supernatant and wash the pellet with 1 ml of 70% EtOH.
5. Centrifuge at 14-16,000 x g for 5 minutes.
6. Completely discard the supernatant and re-suspend the pellets in 50-200 μl of the TE buffer (no provided) or ddH2O.
7. Incubate for 10 minutes at 60°C to dissolve the pellet.
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