Blood DNA Kit Blood genome DNA extraction kit (centrifugal column type)

User Manual

Product Name

Common name: Blood DNA Kit Blood genome DNA extraction kit (centrifugal column type)

Trade name: Blood DNA Kit Blood genome DNA extraction kit (centrifugal column type)

Model: ZT-001
Intended use

This reagent kit uses a centrifugal adsorption column that can specifically bind DNA and a unique buffer system to extract DNA from blood and other samples. The silicon matrix material used in the centrifugal adsorption column can efficiently and specifically adsorb DNA, and combined with a unique buffer system, it can maximize the removal of impurity proteins and other organic compounds in the cell. The extracted genomic DNA fragments are complete, highly purified, and of stable and reliable quality. The purified DNA using this kit is suitable for various conventional experiments such as enzyme digestion, PCR, library construction, Southern hybridization, and experiments that require high-quality DNA such as chip hybridization and high-throughput sequencing.

Inspection principle

1. Wide application of samples: DNA (including genomic DNA, viral DNA, Mitochondrial DNA) can be directly extracted from whole blood (citrate, heparin or EDTA anticoagulation), plasma, serum, white membrane layer, other body fluids, and lymphocytes. 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, the experiment can be completed within 1 hour without the need for phenol chloroform.

4. DNA extraction amount: from 200 μ 1 person can obtain an average of 6 whole blood points μ G DNA (3-12 μ g) . From 200 μ Up to 50 can be obtained from the white membrane layer/5e6 lymphocytes μ G DNA.

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. Extracting samples requires balancing to room temperature.

If there is sediment in the cracking solution 3, it can be dissolved again in a 70 ℃ water bath, shaken well, and then used.

3. All centrifugation steps are performed at room temperature.

4. 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.

5. Balance eluent 3 to room temperature.

Main components

Customers are required to prepare their own items, including: 96~100% ethanol, RNase A (100 mg/ml), PBS, nozzle with filter element, metal bath that can be heated to 58 ° C, centrifuge (capable of placing 1.5ml and 2ml centrifuge tubes), and 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 ℃, the protease and adsorption column 1 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 white membrane layer: fresh whole blood (anticoagulant) is centrifuged at room temperature (15-25 ℃) 2500g for 10 minutes. After centrifugation, it is divided into three parts. The upper transparent layer is plasma, the middle layer is white membrane layer, and the lower layer is red blood cells. Take out the white film layer and follow the following steps to begin the operation.

Note: The white membrane layer is formed after the accumulation of white blood cells in the whole blood. The process of preparing the white membrane layer is very simple, and the DNA yield of the white membrane layer in whole blood is about 5-10 times higher than that of whole blood.

[Operation Steps]

1. Take 1.5ml centrifuge tube and add 25 μ L protease.

2. Add 200 μ L sample (whole blood/plasma/serum/white membrane layer/PBS/other body fluids containing up to 5e6 lymphocytes) and mix well, if the sample is less than 200 μ Fill in with PBS. If you want to remove RNA, you also need to add 4 μ L RNase A (100 mg/ml).

3. Add 200 μ L Cracking liquid 3 is stirred and stirred in a vortex for 15 seconds. Note: Proteinase cannot be directly added to lysate 3.

Note: If the sample size is greater than 200 μ L. The amount of protease and lysate 3 can be increased proportionally; For example, 400 μ For samples, an additional 50 is required μ L protease and 400 μ Cracking liquid 3.

Warm bath at 4.58 ℃ for 10 minutes.

Note: Prolonged warm baths do not affect the yield and quality of extracted DNA.

5. Instantaneous centrifugation, plus 200 μ Mix 96-100% ethanol in a vortex for 15 seconds.

Note: If the room temperature exceeds 25 ° C, cool the ethanol on ice before adding it to the tube. If the sample size is greater than 200 μ L. The amount of ethanol can be increased proportionally by 96-100%; For example, 400 μ For sample l, an additional 400 is required μ 96-100% ethanol.

6. 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. In addition, when the sample is a white membrane layer or PBS containing up to 5e6 lymphocytes, it is recommended to centrifuge at maximum speed to avoid clogging the adsorption column.

7. 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.

8. 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.

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

Replace the centrifuge tube with a new one and add 50 drops to the middle position of the adsorption membrane μ L -200 μ Elute 3, warm bath at room temperature for 1 minute, centrifuge at a rate of 6000g for 1 minute.

Note: A 5-minute warm bath before centrifugation can increase DNA production; If using 200 μ Repetitive elution with eluent 3 will increase DNA recovery (up to a maximum of 15%). For DNA content less than 1 μ G sample, it is recommended to use 50 μ Elution with 3 eluents. Using 2 x 100 μ L replaces 1 × 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.