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uses a unique lysis buffer and proteinase K to digest various types of biological samples to release DNA, and then uses original extraction technology to quickly remove proteolytic products, lipids, polysaccharides, phenolic derivatives, pigments in biological samplesThe specially designed spin column can selectively adsorb large fragments of DNA in the extraction aqueous phase, while small fragments of nucleic acids are selectively filtered and removed. The DNA adsorbed on the spin column only needs to be washed 1 or 2 times with a washing buffer to obtain up to 25 µg of high-purity genomic DNA. Suitable for PCR, Southern blot analysis, RAPD, RFLD and other molecular biology experiments.Rapid Universal Genomic DNA Extraction Kit
Product Composition
Rapid Universal Genomic DNA Extraction Kit Cat. No. | 5 Preps 3105005 | 50 Preps 3 105050 | 250 Preps 3 105250 |
Spin Columns | 5 | 50 | 250 |
2 ml Centrifuge Tubes | 5 | 50 | 250 |
Proteinase K | 120 μl | 1.2ml | 1.2ml×5 |
Buffer GE | 1.8ml | 18ml | 80ml |
Buffer PE | 3ml | 30ml | 130ml |
Buffer WB (concentrate) | 3ml | 19ml | 50ml×2 |
Buffer TE | 3ml | 30ml | 150ml |
Instructions | 1 | 1 | 1 |
1． Proteinase K could be stored at room temperature for at least 1 year. For longer storage, we suggest storing proteinase K at 2-8℃.
2． If other items are stored at room temperature (0~30°C), their performance will remain unchanged within two years; if the product is stored at 2~8°C, the validity period of the product can be extended to more than two years.
This product uses a unique lysis buffer and proteinase K to digest various types of biological samples to release DNA, and then uses original extraction technology to quickly remove proteolytic products, lipids, polysaccharides, phenolic derivatives, pigments in biological samples. and other impurities. The specially designed spin column can selectively adsorb large fragments of DNA in the extraction aqueous phase, while small fragments of nucleic acids are selectively filtered and removed. The DNA adsorbed on the spin column only needs to be washed 1 or 2 times with a washing buffer to obtain up to 25 µg of high-purity genomic DNA. Suitable for PCR, Southern blot analysis, RAPD, RFLD and other molecular biology experiments.
Equipment and Reagents to Be Supplied by User
1． Absolute ethanol
2． 1.5 ml microcentrifuge tubes, pipette, and tips (to avoid contamination between samples, it is recommended to
use pipette tips containing filter elements)
3． Protective equipment such as disposable latex gloves and paper towels
4． Microcentrifuge(s) (with rotor for 1.5 ml and 2 ml microcentrifuge tubes)
5． Water bath and vortexer
6． RNase A (50 mg/ml, Cat. No. 8001001) may be required
1. If the centrifuge has a refrigeration function, please set the temperature to 25°C.
2. Set the water bath temperature to 56 °C and incubate Buffer TE to 56 °C.
3. Add absolute ethanol to Buffer WB according to the instructions on the label of the reagent bottle and check the box on the label to mark "ethanol added".
4. When the room temperature is lower than 15°C, check if precipitates were appeared in Buffer GE and Buffer PE before use. If there is precipitate, dissolve it by incubating at 56°C before use.
【Extraction of genomic DNA from animal tissues】
1. Use a scalpel to cut 20-50 mg of animal tissue, then chop the tissue into a homogenate with the tip of the scalpel and transfer it to a clean 1.5 ml microcentrifuge tube.
* Please add liquid nitrogen to grind the following tissues until they become powdery. Place the mortar in a 56°C water bath. When the powder begins to melt, continue grinding for 1 minute. After homogenization, add 200 μl Buffer TE and 300 μl Buffer GE, continue grinding for 30 seconds and mix, transfer all the homogenate into a clean 1.5 ml microcentrifuge tube, and then continue with step 3. Tissues such as pancreas, spleen, thymus, lymphatic system rich in DNase.
Collagen-rich skin, muscle and other tissues.
Tissues rich in keratin or hard tissues such as bones.
2. Add 150 µl Buffer TE and 300 µl Buffer GE, and vortex for 30 seconds to mix evenly.
3. Add 20 µl Proteinase K and incubate at 56°C water bath for 10 minutes.
* Tissues that have not been ground with liquid nitrogen may still have insoluble particulates present after 10 minutes incubation. This does not affect subsequent operations. If you want to increase the yield of DNA, extend the incubation time until all the tissue is digested before the next step.
4. Add 500 µl Buffer PE, shake vigorously 5 to 10 times to form an emulsion, vortex for 30 seconds to mix evenly, and centrifuge at full speed (≥12,000×g) for 1 minute.
【Extraction of genomic DNA from plant tissues】
1. Weigh an appropriate amount of fresh plant tissue according to the table below (if freeze-dried tissue is used, the amount of tissue should be halved), cut into small pieces and put into a mortar, add liquid nitrogen, and freeze the tissue quickly and firmly grind until powdery. During grinding, liquid nitrogen should be added intermittently to prevent the tissue from melting. After sufficient grinding, place the mortar in a 56°C water bath until the sample powder begins to melt. Add 300 µl Buffer TE and continue grinding for 30 seconds to mix.
Sample type | Recommended dosage |
plant flowers or leaves | 100-200 mg |
Plant roots, stems, seeds | ≤ 240 mg |
* Sample grinding should be sufficient, otherwise the yield of genomic DNA will be seriously affected.
2. Transfer 200 µl of the ground homogenate to a 1.5 ml microcentrifuge tube. If the homogenate volume is less than 200 µl, add Buffer TE to 200 µl.
* If the homogenate is too viscous and cannot be aspirated, it means that too much sample has been used. You can add 200 µl Buffer TE to continue grinding and mixing, and then aspirate 200 µl of the homogenate to continue with next step.
3. Add 300 µl Buffer GE and 20 µl Proteinase K, immediately vortex for 1 minute to mix evenly. Incubate at 56°C water bath for 10 minutes.
* Do not add Proteinase K directly to Buffer GE.
* For samples such as roots/stems rich in fiber or seeds rich in starch and protein, the incubation time can be extended to 30 minutes.
4. Add 500 µl Buffer PE, shake vigorously 5~10 times to form an emulsion, vortex for 30 seconds to mix evenly, and centrifuge at full speed (≥12,000×g) for 5 minutes.
【Extract genomic DNA from cultured cells, lymphocytes, whole blood or bone marrow, and bones】
1a. Suspension cultured animal cells or freshly isolated animal tissue single cell suspension
Centrifuge at 300×g for 5 minutes to collect approximately 5×106 cells. Discard the supernatant and add
200 µl Buffer TE to suspend the cells.
1b. Adherent cultured cells
Discard the culture supernatant, digest and suspend the cells with trypsin, and centrifuge at 300×g for 5 minutes to collect approximately 5×106 cells. Discard the supernatant and add 200 µl Buffer TE to resuspend the cells.
1c. Lymphocytes
Collect about 5×106 lymphocytes and adjust the cell suspension to 200 µl. If the cell suspension is less than 200 µl, add Buffer TE to adjust the final volume to 200 µl.
1d. Blood or bone marrow
Collect 200 µl of anticoagulated whole blood or bone marrow (EDTA anticoagulated).
1e. Bone
Collect 50~100 mg of crushed bone, transfer it into a mortar and grind it into a homogeneous paste; add 300 µl Buffer TE, continue grinding for 30 seconds to mix. Absorb 200 µl of homogenate into a 1.5 ml microcentrifuge tube.
2. Add 300 µl Buffer GE and 20 µl Proteinase K. Immediately vortex for 30 seconds to mix evenly.
* Do not add Proteinase K directly to Buffer GE.
3. Incubate at 56°C water bath for 10 minutes.
4. Add 500 µl Buffer PE, shake vigorously 5~10 times to form an emulsion, vortex for 30 seconds to mix
evenly, and centrifuge at full speed (≥12,000×g) for 1 minute.
【Extracting genomic DNA from bacteria】
* . Collect 3~5 ml bacterial culture in a 1.5 ml microcentrifuge tube, add 100 μl Buffer TE, and vortex to fully suspend the bacteria. Add 100 μl of lysozyme solution (Cat. No. 8009500, prepared by the user), vortex for about 15 seconds to mix, and incubate in a 37°C water bath for 30 minutes.
* Certain divalent cations will inhibit the activity of lysozyme. If the bacterial culture medium contains divalent cations (such as MRS culture medium, etc.), a washing step should be added after centrifuging to collect the bacteria: add 1 ml of deionized pure water and vortex After shaking to suspend the bacteria, centrifuge at 12,000 ×g for 30 seconds, discard the ionized pure water, then add 100 μl Buffer TE, and vortex to fully suspend the bacteria.
* Preparation method of lysozyme solution: Add 1 ml of deionized pure water for every 100 mg of lysozyme to prepare a 100 mg/ml lysozyme solution.
* Freezing and thawing of lysozyme solution will seriously reduce the lysis efficiency. Please try to use freshly prepared lysozyme solution or freeze and thaw no more than once.
2. Add 300 µl Buffer GE and 20 µl Proteinase K. Immediately vortex for 30 seconds to mix evenly.
* Do not add Proteinase K directly to Buffer GE.
3. Incubate at 56°C water bath for 10 minutes.
4. Add 500 µl Buffer PE, shake vigorously 5~10 times to form an emulsion, vortex for 30 seconds to mix
evenly, and centrifuge at full speed (≥12,000×g) for 1 minute.
【Extraction of genomic DNA from yeast】
* . Collect 5~10 ml of yeast cultured overnight and discard the supernatant. Add 100 µl Buffer TE, do not discard the pipette tip, pipe the yeast pellet directly with the pipette tip, suck it out and transfer it to the mortar. Pour in liquid nitrogen to submerge the yeast slurry (the yeast slurry will immediately condense into clumps when it hits by the liquid nitrogen), and grind vigorously until the yeast clumps become powdery.
* If you use a 1.5 ml microcentrifuge tube to collect yeast, you can centrifuge at 12,000 rpm for 30 seconds multiple times to enrich the yeast.
* If the yeast clumps cannot be ground into powder, liquid nitrogen should be added to continue grinding, otherwise the final DNA recovery efficiency will be seriously affected.
* In the absence of liquid nitrogen, direct grinding can be used for about 15 minutes to fully destroy the cell wall. If the liquid is evaporated during this process, add 100 µl Buffer TE and continue grinding.
2. When the powdered yeast begins to melt, add 100 µl Buffer TE and 2 µl RNase A (Cat. No. 8001001, user- prepared) to the mortar, continue grinding several times, and transfer the lysate to 1.5 ml microcentrifuge tube, Incubate at 56°C water bath for 5 minutes.
* If the lysate is less than 200 µl, add Buffer TE to 200 µl.
3. Add 300 µl Buffer GE and 20 µl Proteinase K, vortex for 30 seconds to mix, incubate at 56°C water bath for 20 minutes. During the incubation, turn the centrifuge tube several times every 2~3 minutes to help release the DNA.
* Do not add Proteinase K directly to Buffer GE.
4. Add 500 µl Buffer PE, shake vigorously 5~10 times to form an emulsion, vortex for 30 seconds to mix evenly, and centrifuge at full speed (≥12,000×g) for 1 minute.
【Extraction of genomic DNA from stool】
1. Use 1.5 ml microcentrifuge tube to collect 30~50 mg stool, add 150 µl Buffer TE, vortex until all particles are suspended.
* If the sample is liquid, pipet 200 µl of sample; if DNA is extracted from mouse feces, add 150 µl of Buffer TE and then grind the fecal particles with a grinding rod (Cat. No. D-050, provided by the user).
2. Add 300 µl Buffer GE and 20 µl Proteinase K. Immediately vortex for 30 seconds to mix evenly.
* Do not add Proteinase K directly to Buffer GE.
3. Incubate at 56°C water bath for 10 minutes.
4. Add 500 µl Buffer PE, shake vigorously 5~10 times to form an emulsion, vortex for 30 seconds to mix evenly, and centrifuge at full speed (≥12,000×g) for 1 minute.
5. Aspirate all the supernatant and add it to a spin column (the spin column is placed at 2 ml collection tube), cap the tube, and centrifuge at 12,000×g for 30 seconds.
* It is better to aspirate less supernatant than to aspirate the interphase precipitate, which will seriously affect the purity of the final DNA.
6. Discard the filtrate and place the spin column back to the 2 ml collection tube, add 800 µl Buffer WB to the spin column, centrifuge at 12,000×g for 30 seconds.
* There is no need to discard the filtrate completely. If you want to avoid contamination of the centrifuge by the filtrate adhering to the mouth of the centrifuge lid, place the 2 ml collection tube upside down on a paper towel and tap it once.
* Ensure that absolute ethanol has been added to Buffer WB.
7. Discard the filtrate and place the spin column to the 2 ml collection tube, add 300 µl Buffer WB to the spin column, centrifuge at full speed(≥12,000×g) for 1 minute.
8. Discard the 2 ml collection tube and filtrate, place the spin column in a clean 1.5 ml microcentrifuge tube, add 100~200 µl Buffer TE incubated at 56°C to the center of the column membrane, close the lid, and let stand at room temperature for 1 minute. Centrifuge at 12,000 ×g for 30 seconds.
* Be careful not to let the filtrate touch the bottom of the spin column when taking out the spin column. If the spin column is contaminated with filtrate, please discard the filtrate, and put the spin column back into the 2 ml collection tube and centrifuge at full speed for 1 minute before taking it out, then continue with this step.
* If the centrifuge does not have a leak-proof cover, please change the centrifugation conditions to 8,000 ×g for 1 minute to prevent the tube lid from falling off and damaging the centrifuge.
9. Discard the spin column and the eluted DNA can be used immediately for various molecular biology experiments; or the DNA can be stored at -20 ℃ for later use.
