Blood DNA Kit Tissue Genome DNA extraction Kit (column type)

User Manual

Product Name

Common name: Blood DNA Kit Tissue Genome DNA extraction Kit (column extraction type)

Trade name: Blood DNA Kit Tissue Genome DNA extraction Kit (column extraction type)

Model: ZT-006
Intended use

This kit uses a centrifugal adsorption column that can specifically bind nucleic acids and a unique buffer system to extract nucleic acids from tissue samples. The silicon matrix material used in the centrifugal adsorption column can efficiently and specifically adsorb nucleic acids, and combined with a unique buffer system, it can maximally remove impurities such as proteins and other organic compounds in cells. The extracted nucleic acid fragments are complete, highly purified, and of stable and reliable quality. The nucleic acid purified using this kit is suitable for various conventional experiments such as enzyme digestion, PCR, library construction, Southern hybridization, and experiments that require high-quality nucleic acid such as chip hybridization and high-throughput sequencing. This product is expected to be used by professionals, such as technicians and doctors trained in molecular biology technology.

Inspection principle

1. Samples: DNA (including genomic DNA and Mitochondrial DNA) can be directly extracted from tissues, and samples can be fresh or frozen.

2. High quality: Unique lysis buffer system, purified DNA with high concentration, high purity, and good integrity, meeting experimental requirements such as chip hybridization and high-throughput sequencing.

3. Fast and non-toxic: Adopting the principle of silica gel membrane adsorption, there is no need for phenol chloroform.

Precautions (please be sure to read this note before using this reagent kit):

1. The sample should avoid repeated freeze-thaw, otherwise it may result in smaller extracted DNA fragments and a decrease in extraction volume.

If there are precipitates in cracking solution 1 and 3, they can be dissolved again in a 70 ℃ water bath, shaken well, and then used.

3. RNA can inhibit some downstream enzyme reactions, but it will not inhibit PCR. To remove RNA residues, you need to prepare your own RNase A (100 mg/ml) solution.

4. Balance eluent 3 to room temperature.

5. All centrifugation steps are performed at room temperature.

6. Prepare tissue samples on a cold surface (such as a glass plate, steel plate, or aluminum plate placed above dry ice).

7. If frozen tissue is used, ensure that the frozen sample does not dissolve before adding lysate 1.

【 Main components 】

Customers are required to prepare their own items, including: 96~100% ethanol, RNase A (100 mg/ml), nozzle with filter element, metal bath that can be heated to 58 ° C/70 ° C and mixed evenly, centrifuge (capable of placing 1.5ml and 2ml centrifuge tubes), vortex oscillator.

Storage conditions and validity period

The reagent kit can be stored for 12 months under dry conditions at room temperature (15-25 ℃). If the room temperature exceeds 25 ℃, adsorption column 1 and protease can be stored at 2-8 ℃.

Reagent preparation

Before use, add 96-100% ethanol to Lotion 3 and Lotion 4, and add 17ml 96-100% ethanol to Lotion 3 bottles; Add 21ml of 96-100% ethanol to 4 bottles of washing solution. Add 1.5ml of protease solution to the protease bottle for dissolution (store the dissolved protease at 2-8 ℃).

Sample Preparation

Preparation of tissue samples (fresh or frozen):

1. Fresh tissue samples can be cut into small pieces and stored at -20 ℃ or -80 ℃.

2. Take a certain amount of tissue sample (about 25mg, spleen tissue about 10mg), and choose one of the following three methods for treatment (b, c, these two methods will take less than a sample lysis time): a. Cut the tissue into small pieces and add 220 μ Cracking liquid 1 and 25 μ L protease; b. Place the tissue in liquid nitrogen and thoroughly grind with a mortar and pestle. Then pour the tissue powder and liquid nitrogen into a 1.5 milliliter microcentrifuge tube. Allow liquid nitrogen to evaporate (but do not allow tissue to thaw) and add 220 μ Cracking liquid 1 and 25 μ L protease; c. Organize and 80 μ Add PBS to the homogenizer for homogenization, and after tissue homogenization, add 140 μ Cracking liquid 1 and 25 μ L protease.

Note: Some organizations require undiluted lysis solution 1 to fully cleave, and in this case, it is recommended to use method b; The yield of DNA depends on the number and type of tissues treated. A milligram of tissue will produce approximately 0.2-1.2 micrograms of DNA.

3. Then place it in a whirlpool shakeable warm bath at 58 ℃ and mix at 900rpm until the tissue is completely dissolved (in most cases, it can be completely dissolved within 3 hours, and sometimes overnight lysis may be necessary to obtain the best results).

4. Instantaneous centrifugation, adding 4 μ 1. RNase A (100 mg/ml) at room temperature (15-25 ℃) for 2 minutes; If RNA removal is not necessary, skip to the next step without doing so.

5. If insoluble substances still exist, centrifuge 3000g for 1 minute and extract nucleic acid from the supernatant.

[Operation Steps]

1. Add 245 to the above supernatant μ L cracking solution 3, thoroughly mix, and soak at 70 ℃ for 10 minutes.

2. Instantaneous centrifugation, adding 245 μ Ethanol (96% -100%), cover, vortex oscillate and mix for 15 seconds. Incubate at room temperature (15-25 ° C) for 5 minutes.

Note: If the room temperature exceeds 25 ° C, cool the ethanol on ice before adding it to the tube.

3. Instantaneous centrifugation, add to the adsorption column, centrifuge at a speed of over 6000g for 1 minute, place the adsorption column in a clean 2 ml collection tube, and discard the collection tube containing the effluent. Note: 6000g centrifugation will only reduce noise, and centrifugation at a higher speed will not affect DNA extraction.

4. Add 500 μ Centrifuge at a rate of 6000g of washing solution 3 (with ethanol added) for 1 minute, place the adsorption column in a clean 2 ml collection tube, and discard the collection tube containing the effluent.

5. Add 500 μ Centrifuge solution 4 (with ethanol added) at a rate of 20000g for 3 minutes, place the adsorption column in a clean 2 ml collection tube, and discard the collection tube containing the effluent.

6. Suggested steps: Centrifuge at a speed of 20000g for 1 minute.

7. Replace the centrifuge tube and add 200 drops to the middle position of the adsorption membrane μ Elute 3, warm bath at room temperature for 1 minute, centrifuge at a rate of 6000g for 1 minute.

8. Repeat step 7.

Note: A 5-minute warm bath before centrifugation can increase DNA production; If using 200 μ Perform the third elution with eluent 3, which will increase DNA recovery by up to 15%. Using 4x 100 μ L replaces 2 × two hundred μ L elution does not increase elution efficiency. If the extracted product requires long-term storage, it is recommended to store it at -30 ° C to -15 ° C.