Blood DNA Kit DNA extraction kit for cultured cells (centrifugal column type)

User Manual

Product Name

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

Trade name: Blood DNA Kit DNA extraction kit for cultured cells (centrifugal column type)
Model: ZT-009

Intended use

This kit uses a centrifugal adsorption column that can specifically bind DNA and a unique buffer system to extract DNA from cultured cells. 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. DNA (including genomic DNA, viral DNA, Mitochondrial DNA) can be directly extracted from cultured cells, 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, the experiment can be completed within 1 hour without the need for phenol chloroform.

4. Amount of DNA extracted: 50% can be obtained at most from 5e6 cultured cells (with normal genome) 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. Balance eluent 3 to room temperature.

【 Main components 】

Customers need to prepare their own items, including: 96~100% ethanol, PBS, gun head with filter element, metal bath that can be heated to 58 ℃, 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

1. Cell collection method 1: After determining the number of suspended growing cells (no more than 5e6 cells), centrifuge 300g in a 1.5ml centrifuge tube for 5 minutes, remove the supernatant, and be careful not to disturb cell sedimentation.

2. Cell collection method 2: Monolayer grown cells are digested with trypsin or separated from the culture using a cell scraper. Trypsin digestion of cells: After determining the number of cells. Extract the culture medium and wash the cells with PBS. Then extract PBS and add 0.10-0.25% trypsin. After the cells detach, collect them and transfer an appropriate number of cells (no more than 5e6 cells) to a 1.5ml centrifuge tube. Centrifuge at 300g for 5 minutes. Remove the supernatant and be careful not to disturb cell sedimentation.

3. Use cell scraper: take out cells from Petri dish or flask. Transfer an appropriate number of cells (not exceeding 5e6 cells) to a 1.5ml centrifuge tube and centrifuge at 300g for 5 minutes. Remove the supernatant and be careful not to disturb cell sedimentation.

4. Choose one of the two cell collection methods mentioned above to collect cells and resuspend at 200 μ 1PBS.

5. Add 25 μ Mix the protease well. Then start with the extraction steps below.

[Operation Steps]

1. 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 2.58 ℃ for 10 minutes. Note: Prolonged warm baths do not affect the yield and quality of extracted DNA.

3. 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.

4. 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.

5. 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.

6. 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.

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

8. Replace the centrifuge tube and add 50 drops to the middle position of the adsorption membrane μ L -200 μ Elute 3, warm bath at room temperature for 5 minutes, centrifuge at a rate of 6000g for 1 minute. Note: 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.