Sample4 rxns ($19.2)
Sample4 rxns ($19.2)
Note: PureDireX / Purification Kits / Extraction Kit / Plant
The Genomic DNA Isolation Kit (Plant) is designed specifically for genomic DNA isolation from plant samples. This unique buffer system ensures total DNA with high yield and good quality from samples. The spin column system is designed to purify or concentrate DNA products which have been previously isolated using buffers. The entire procedure can be completed in 1 hour without phenol/chloroform extraction. Purified DNA is suitable for use in PCR or other enzymatic reactions.
|Buffer PL||55 ml||2 ml|
|Buffer W1||45 ml||2 ml|
|Buffer W2 (Add ethanol)||15 ml (60 ml)||300 µl × 2 (1.5 ml × 2)|
|Buffer E||10 ml||1 ml|
|Column PC||100 pcs||4 pcs|
|Collection Tubes||100 pcs||4 pcs|
1. Cut off 50 mg of the fresh plant tissue or 25 mg of the dry plant tissue.
2. Grind the sample under liquid nitrogen to a fine powder using a mortar and pestle.
1. Add 500 µl of the Buffer PL 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 75°C for 30 minutes. (invert the tube every 10 minutes)
4. Centrifuge at 14-16,000 x g for 5 minutes.
5. Transfer the supernatant to a new 1.5 ml microcentrifuge tube. #Pre-heat the Buffer BE to 75°C for Step 5 DNA Elution.
1. Add the same volume of the isopropanol to the clear supernatant from the previous step and vortex immediately for 5 seconds (eg. add 350 µl Isopropanol to the 350 µl supernatant)
2. Place a Column PC in a 2 ml Collection Tube.
3. Transfer the mixture to the Column PC.
4. Centrifuge at 14,000 x g for 30 seconds.
5. Discard the flow-through and place the Column PC back in the same Collection Tube.
1. Add 400 µl of the Buffer W1 into the Column PC.
2. Centrifuge at 14,000 x g for 30 seconds.
3. Discard the flow-through and place the Column PC back into the same Collection tube.
4. Add 600 µl of the Buffer W2 (Ethanol added) into the Column PC.
5. Centrifuge at 14,000 x g for 30 seconds.
6. Discard the flow-through and place the Column PC back into the same Collection tube.
7. Centrifuge at 14,000 x g again for 2 minutes to remove the residual Buffer W2.
1. Transfer the dried Column PC to a new 1.5 ml microcentrifuge tube.
2. Add 50-200 µl of the Pre-Heated Buffer BE or TE into the center of the column matrix.
3. Let stand at 75°C for 3 minutes.
4. Centrifuge for 2 minutes at 14,000 x g to elute the purified DNA.
Refer to the table below to troubleshoot problems that you may encounter when purifying the genomic DNA with the kit.
|Low yield of DNA||Incomplete lysed sample||Use the appropriate method for the lysate preparation based on the amount of the starting materials.|
|Increase the digestion time.|
|DNA degrade||Sample not fresh||Avoid repeated freeze / thaw cycles of the sample.|
|Use a new sample for the DNA isolation. Perform the extraction of the fresh material when possible.|
|Inappropriate sample storage conditions||Store culture cell / fungus at -20°C until use. The whole blood can be stored at 4°C for no longer than 1-2 days.|
|DNase contaminant||Use the fresh TAE or TBE electrophoresis buffer.|
|Maintain a sterile work environment to avoid contamination from DNases.|
|Presence of RNA||RNA contamination||Perform RNase A digestion step during Step Lysis.|
|Inhibition of downstream enzymatic reactions||Presence of ethanol in purified DNA||Discard the ethanol of the Buffer W2 flow through from the collection tube. Place the spin cartridge into the collection tube and centrifuge the spin cartridge at maximum speed for 2–3 minutes to completely dry the cartridge.|
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