Animal Tissue Genomic DNA Extraction Kit Instruction Manual

Animal tissue genomic DNA extraction kit

First, the introduction

The animal tissue genomic DNA extraction kit (DePure Tissue DNA Kit) is suitable for DNA extraction of biological samples such as animal tissues, cultured cells, anticoagulant, body fluids, and secretions. The kit is based on silica gel column purification technology. It does not require the use of toxic phenol chloroform extraction during extraction, and does not require time-consuming alcohol precipitation. The entire extraction process takes only 30 minutes (excluding digestion time). The animal tissue genomic DNA extraction kit can process 1~20mg tissue samples, 1~200μl anticoagulant or body fluid samples, and 5×10 ⁶ culture cells at one time. The obtained DNA can be directly used for PCR, Southern blot, LAMP, Real- Downstream experiments such as Time PCR.

Second, the principle

The animal tissue genomic DNA extraction kit is based on a silica gel column purification method. The sample was lysed by lysis buffer (Buffer MTL) and protease, and the DNA was released into the lysate. After adding Buffer AL and ethanol, it is transferred to a column for filtration, and the DNA is adsorbed onto the membrane of the column, while the protein is not adsorbed and is removed by filtration with the solution. The column is washed with Buffer GW1 for protein and other impurities, washed by Buffer GW2 to remove salt. Finally, the DNA is eluted by low salt buffer (Buffer AE). The eluted DNA can be directly used for PCR, Southern blot, LAMP, Real-Time PCR. Wait for downstream experiments.

The DePure silica gel column is based on a high binding glass fiber filter. The filter membrane can adsorb nucleic acids by physical interactions such as hydrogen bonding and static electricity under the condition of high concentration ionizing agent (such as guanidine hydrochloride or guanidinium isothiocyanate), and proteins or other impurities are not adsorbed and removed. The nucleic acid-adsorbed filter is washed to remove proteins and salts, and finally the nucleic acid adsorbed on the filter can be eluted with a low salt buffer (Buffer AE). The obtained nucleic acid is high in purity and can be directly used in various downstream experiments.

Third, the composition

DePure Tissue DNA Kit

Fourth, the shelf life

The animal tissue genomic DNA extraction kit can be dried for 12 months at room temperature (15-25 ° C) except Proteinase K. It should be placed at 2-8 °C for long-term storage. At low temperatures, Buffer MTL may form precipitates, requiring a 37 ° C water bath to completely dissolve the precipitate. Proteinase K dry powder and RNase Solution are transported at room temperature. Store at 2~8°C or -20°C after receiving the kit .

V. Need to prepare materials and tools

• Anhydrous ethanol (96%-100%)
• Sterilized centrifuge tubes and tips
• Small centrifuge (13,000 rpm)
• Water bath or metal bath
• Add the appropriate amount of Protease Dissolve Buffer to the Proteinase K dry powder to a final concentration of 20 mg/ml as indicated on the bottle label. Mix inversion and allow the proteinase K to dissolve fully and store at -20 °C.

• Before using Buffer GW1, it should be diluted with absolute ethanol as indicated on the bottle label.

• Buffer GW2 should be diluted with absolute ethanol as indicated on the bottle label before use.

6. Scheme 1. DNA extraction from animal tissues

The protocol is suitable for extracting DNA from tissue samples of 1-20 mg animal tissues such as liver, spleen, kidney, mouse tail, etc. The following centrifugation operations are performed at room temperature.

• Cut 1 to 20 mg of tissue sample (or 10 mg of liver, lung or spleen) into as small pieces as possible and transfer to a 1.5 ml centrifuge tube. Add 230 μl Buffer MTL and 20 μl Proteinase K and vortex to mix. The sample was digested in a 55 ° C water bath for 1-3 hours or overnight. Occasionally vortex and mix during the water bath, or place in the oscillating water bath vortex.

Note: Appropriate tissue usage can achieve the desired extraction results, too much sample will reduce yield and purity. Tissue samples such as the spleen, liver, and kidney are rich in DNA and should not exceed 10 mg. Samples such as muscles and skin can be used up to 30 mg to increase yield. Cutting the tissue block into small pieces as much as possible can shorten the digestion time. Using liquid nitrogen grinding, the homogenizer can treat tissue samples to shorten the digestion time. The maximum amount of mouse tail is 1.2 cm, and the maximum amount of rat tail is 0.6 cm. The digestion time depends on the type of sample and the homogenization effect. It takes 1-3 hours for a typical tissue sample and 6-8 hours for a rat tail. There was no negative impact on overnight digestion.

• Add 5 μl of RNase Solution to the digestive juice and mix. Degrade RNA by placing it in a water bath at room temperature or 37 ° C for 15 to 60 minutes.

Note: The RNase Solution digestion time depends on the sample type. The liver and kidney are rich in RNA and require longer digestion times.

• (Optional) Centrifuge at 10,000 rpm for 3 minutes. Transfer the supernatant to a new 1.5 ml centrifuge tube.

Note: If the digestive juice is cloudy or there are obvious particles, do not omit this step.

• Add 250 μl Buffer AL to the digestive juice. Mix the highest speed vortex for 30 seconds. Water bath at 70 ° C for 10 minutes.
• 250 μl of absolute ethanol was added to the digestive juice. Mix the highest speed vortex for 30 seconds.

Note: When treating tissues such as the liver or spleen, precipitation occurs when ethanol is added, which is normal. Use a pipette to suck 10-15 times to break up the pellet as much as possible.

• The DePure gDNA Mini Column was placed in a 2 ml collection tube. Transfer the mixture obtained in the fifth step (including precipitation) to the column. Centrifuge at 10,000 rpm for 1 minute.

Note: If the column is clogged, extend the centrifugation time.

• Discard the effluent and reinstall the column back into the collection tube. Add 500 μl Buffer GW1 (diluted with ethanol) to the column. Centrifuge at 10,000 rpm for 1 minute.

Note: Buffer GW1 must be diluted with absolute ethanol. Dilute according to the bottle label or instructions.

• The filtrate was discarded and the column was reloaded back into the collection tube. Add 500 μl Buffer GW2 (diluted with ethanol) to the column. Centrifuge at 10,000 rpm for 1 minute.

Note: Buffer GW2 must be diluted with absolute ethanol. Dilute according to the bottle label or instructions.

• The filtrate was discarded and the column was reloaded back into the collection tube. Add 500 μl Buffer GW2 (diluted with ethanol) to the column. Centrifuge at 10,000 rpm for 1 minute.
• Discard the effluent and reinstall the column back into the collection tube. The residual ethanol in the column was removed by centrifugation at 10,000 × g for 2 minutes.
• The column was placed in a new 1.5 ml centrifuge tube. Add 30~100μl to preheat to 70°C Buffer AE to the center of the column. Leave for 3 minutes. Centrifuge at 10,000 rpm for 1 minute.
• (Optional) Add 30~100μl to preheat to 70°C Buffer AE to the center of the column. Leave for 3 minutes. Centrifuge at 10,000 × g for 1 minute.

Note: Samples with very low DNA content do not require a second elution.

• Discard the DNA binding column, store the DNA at 2-8 ° C, and store it at -20 ° C for long-term storage.

Seven, program 2. Body fluid / cell DNA extraction

The protocol is suitable for direct extraction of DNA from animal blood, serum, plasma, saliva and other liquid samples, and the following centrifugation is performed at room temperature.

• Sample processing
  • Blood samples with red blood cells with nucleus: Red blood cells with nuclei in the blood of non-mammals such as birds/fish have extremely rich DNA content. In a 1.5 ml centrifuge tube, 1-10 μl of anticoagulant sample was added. Adjust the total volume to 200 μl with Buffer PBS or sterile water, vortex and mix, and proceed to step 2.
  • Blood samples without red blood cells in erythrocytes: 1-200 μl anticoagulant sample in a 1.5 ml centrifuge tube. Adjust the total volume to 200 μl with Buffer PBS or sterile water and follow step 2.
  • Saliva or other liquid sample: In a 1.5 ml centrifuge tube, add 1-200 μl saliva or other liquid sample, adjust the total volume to 200 μl with Buffer PBS or sterile water, proceed as in step 2.
  • Cultured cells (5 x 10 ⁶ ): The cells were collected by centrifugation at 500 rpm, and the culture solution was discarded. Resuspend the cells by vortexing with 200 μl Buffer PBS or sterile water.
• Add 20 μl of Proteinase K to the sample and mix gently by tapping several times.
• Add 200 μl Buffer AL to the sample and vortex for 30 seconds at the highest speed. 65 ° C water bath for 30 minutes.

Note: To remove RNA, add 5ul of RNase A to the digestive juice and let stand for 15 to 30 minutes at room temperature.

• 200 μl of absolute ethanol was added to the digestive juice. Mix the highest speed vortex for 30 seconds.

Note: If a precipitate forms after the addition of ethanol, use a pipette to pump 5 times to break up the pellet.

• Follow steps 6 through 13 of scenario 1.

Eight, frequently asked questions

This list may help you solve the problems you encountered during the extraction process. If you have questions or have any suggestions for the kit, or if you have problems with molecular biology experiments, please contact us. We will do our best to help you solve problems.

If the above solution still does not solve your problem, please contact us.

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