Description
This kit is suitable for rapid and simple extraction of genomic DNA from various blood/cell/tissue samples. The extracted genomic DNA features large fragment size, high purity, and good stability, and can be directly used in experiments such as PCR, enzyme digestion, and hybridization. The extraction process does not require phenol-chloroform extraction: blood, cells, or tissues are lysed with lysis buffer and digested with Proteinase K, then can bind to Spin Columns GH4 under the adjustment of absolute ethanol. Residual impurities such as proteins and salts are removed through rapid and thorough washing, and finally, DNA is eluted in Buffer EB.Product Components and Storage Conditions:M665559Component50TStorageM665559ABuffer DA15 mlRTM665559BBuffer DLB15 mlRTM665559CBuffer DW113 mlRTM665559DBuffer DW2Plus12 mlRTM665559EBuffer EB15 mlRTM665559FProteinase K（20 mg/ml）1.2 mlRTM665559GSpin Columns GH450 pcsRTM665559H2 ml Collection Tubes50 pcsRTStorage Conditions:All components of this kit can be stored at room temperature (15°C-25°C) under dry conditions for 12 months. Before use, check if Buffer DA and Buffer DLB have crystals or precipitates. If crystals or precipitates are present, place Buffer DA and Buffer DLB in a 37°C water bath to redissolve.Precautions:1. Samples should avoid repeated freezing and thawing, as this will result in smaller extracted DNA fragments and reduced extraction yield.2. Before first use, 17mL of absolute ethanol must be added to Buffer DW1 (13mL), and 48mL of absolute ethanol must be added to Buffer DW2Plus (12mL).3. If RNA removal is required, RNase A (100 mg/ml) (Catalog No.: R665518) must be prepared separately.4. Absolute ethanol must be prepared separately.Operating Steps:Before use, add absolute ethanol to Buffer DW1 and Buffer DW2Plus. Please refer to the labels on the bottles for the volume to be added.1. Sample Processing:a. Mammalian blood: Take 200 µl of fresh or anticoagulated blood into a 1.5 ml centrifuge tube, and proceed directly to steps 2-10. If the blood volume is less than 200 µl, make up to 200 µl with Buffer DA.b. Avian, poultry, and amphibian blood: Take 5-20 µl of fresh or anticoagulated blood into a 1.5 ml centrifuge tube, and make up to 200 µl with Buffer DA.c. Tissue culture cells: Collect approximately 10⁵-10⁶ suspension cells (total cell count should not exceed 5×10⁶) into a 1.5 ml centrifuge tube; for adherent cells, first digest with trypsin and resuspend by pipetting. Centrifuge at 12,000 rpm (~13,400×g) for 1 min, discard the supernatant, add 200 µl Buffer DA to the cell pellet, and vortex until completely resuspended.d. Animal tissue: Weigh < 25 mg of animal tissue (spleen should be less than 10 mg), grind into fine powder with liquid nitrogen or cut into small pieces with a scalpel, and transfer to a 1.5 ml centrifuge tube pre-filled with 180 µl Buffer DA.In addition to efficiently extracting DNA from blood, cells, and tissues, this kit can also extract high-quality DNA from bacteria.e. Bacteria: Take 1-5 ml of bacterial culture, centrifuge at 12,000 rpm (~13,400×g) for 1 min, and aspirate the supernatant as much as possible.1)For gram-positive bacteria with difficult cell wall lysis, add lysozyme solution for cell wall disruption: Add 110 µl buffer (20 mM Tris, pH 8.0; 2 mM Na₂-EDTA; 1.2% Triton) and 70 µl lysozyme solution (50 mg/ml, provided by the user), and incubate at 37°C for at least 30 min.2)For relatively easily lysed bacteria, directly add 200 µl Buffer DA to the bacterial pellet and vortex until the bacteria are completely resuspended.2. Optional step: If RNA removal is required (for cells, tissues, and bacteria), add 4 µl RNase A (100 mg/ml) solution (Catalog No.: R665518). Vortex for 15 sec and incubate at room temperature for 5 min.3. Add 20 µl Proteinase K and vortex to mix thoroughly.a. For blood, cell, or bacterial samples, simply add Proteinase K, mix well, and proceed to the next step.b. For animal tissue samples, add Proteinase K and vortex to mix. Incubate at 56°C until the tissue is completely dissolved, then briefly centrifuge to collect the solution on the inner wall of the tube cap before proceeding to the next step.Note: Lysis time varies for different tissues, usually taking 1-3 hours (mouse tail requires 6-8 hours of digestion, and overnight digestion is necessary if needed), which will not affect subsequent operations.4. Add 200 µl Buffer DLB, invert thoroughly to mix, incubate at 70°C for 10 min, and briefly centrifuge to collect the solution on the inner wall of the tube cap.Note: White precipitate may form when adding Buffer DLB, but it usually disappears during incubation at 70°C and will not affect subsequent experiments.5. Add 200 µl absolute ethanol and mix (precipitate may appear at this point). Transfer the resulting solution and precipitate to the adsorption column Spin Columns GH4 (place the column in the collection tube), centrifuge at 12,000 rpm (~13,400×g) for 30 sec, discard the filtrate, and return the adsorption column to the collection tube.6. Add 500 µl Buffer DW1 (ensure absolute ethanol is added before use) to the adsorption column, centrifuge at 12,000 rpm (~13,400×g) for 30 sec, discard the waste liquid, and return the adsorption column to the collection tube.7. Add 500 µl Buffer DW2Plus (ensure absolute ethanol is added before use) to the adsorption column, centrifuge at 12,000 rpm (~13,400×g) for 30 sec, discard the waste liquid, and return the adsorption column to the collection tube.8. Repeat step 7 once.9. Centrifuge at 12,000 rpm for 3 min, and discard the waste liquid in the collection tube.10. Transfer the adsorption column to a new 1.5 ml centrifuge tube, 悬空滴加 60-200 µl Buffer EB to the center of the adsorption membrane, let stand at room temperature for 1 min, and centrifuge at 12,000 rpm (~13,400×g) for 1 min to obtain the DNA solution.Note: The volume of Buffer EB should not be less than 60 µl, as a smaller volume will affect recovery efficiency. If water is used as the eluent, ensure its pH is within the range of 7.0-8.5 (NaOH can be used to adjust the pH of water to this range). Store the DNA solution at -20°C.Frequently Asked Questions and Solutions:Frequently Asked QuestionsCauseSolutionColumn blockage1. Too much sample inputPlease use samples within the compatible range.2. Inadequate Sample LysisExtend the 56°C water bath time and increase the number of inversion and mixing times.Low DNA Yield1. The sample has been repeatedly frozen and thawedAvoid repeated freezing and thawing of samples. It is recommended to use fresh samples or samples that have been frozen frozen and thawed only once.2. Inadequate lysis of animal tissueCut tissue blocks into small pieces as much as possible or grind them with liquid nitrogen. During extraction, ensure that the samples are fully mixed with Buffer DA and Proteinase K in a timely manner, and the 56°C water bath lysis time can be appropriately extended.3. Incomplete transfer of the lysis mixture into the adsorption columnAfter cell lysis, add absolute ethanol, flocculent precipitates will appear in the solution, which need to be transferred to the adsorption column together with the solution.4. Issues with the eluentPlease use the Buffer EB provided with the kit for elution. If using ddH₂O or other eluents, ensure that the pH value of the eluent is between 7.0 - 8.0. Buffer EB can be preheated to 55°C in advance before elution, which is beneficial to improve the DNA yield.5. Low elution efficiencyThe eluent should be dropped in the center of the membrane; increase the elution volume or the number of elutions.6. Failure to add absolute ethanol to Buffer DW1/DW2PlusAdd the specified volume of absolute ethanol to Buffer DW1 and Buffer DW2Plus as indicated on the bottle labels.Low DNA Purity1. Contamination with foreign proteinsBuffer DW1 was not used for rinsing, or the correct volume of absolute ethanol was not added to Buffer DW1. Please add the specified volume of absolute ethanol according to the bottle label before operation.2. Contamination with impurity ionsBuffer DW2Plus was not used for rinsing, or rinsed only once. Please rinse twice with Buffer DW2Plus according to the instruction manual