Blood DNA Kit Bacterial DNA extraction kit (column type)

User Manual

Product Name

Common name: Blood DNA Kit Bacterial DNA extraction Kit (column extraction type)

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

Model: ZT-005
Intended use

This reagent kit uses a centrifugal adsorption column that can specifically bind nucleic acids and a unique buffer system to extract nucleic acids from the sample. 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. Wide application of samples: DNA can be extracted from Escherichia coli, Bacillus subtilis, Bordetella pertussis from Nasopharyngeal swab, Borrelia burgdorferi from cerebrospinal fluid, Legionella pneumophila pneumophila from alveolar lavage fluid and Gram-positive bacteria.

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. Balance the sample and eluent 3 to room temperature.

4. All centrifugation steps are performed at room temperature.

【 Main components 】

Items that customers need to prepare by themselves include: 96~100% ethanol, RNase A (100 mg/ml), PBS containing ordinary bactericide for swab samples, 20 mg/ml Lysozyme or 200 mg/ml Lysozyme for Gram-positive bacteria and bacteria difficult to lyse μ G/ml lysostaphin (soluble in 20 mM Tris · HCl, pH 8.0; 2 mM EDTA; 1.2% Triton X-100), a metal bath with a filter gun head, capable of heating to 58 ° C/95 ° C and mixing well, a centrifuge (capable of placing 1.5ml and 2ml centrifuge tubes), and a 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

1. Bacterial DNA in biological fluid: Centrifuge 5000g for 10 minutes and resuspend the precipitate at 180 ° C μ Cracking liquid 1. Then extract according to the nucleic acid extraction steps.

2. Isolate bacterial DNA from eyes, nose, pharynx or other swabs: collect samples, put them into 2ml PBS containing ordinary Fungicide, and incubate them at room temperature (15-25 ℃) for several hours; Centrifuge 5000g for 10 minutes and resuspend the precipitate at 180 ° C μ Cracking liquid 1. Then extract according to the nucleic acid extraction steps.

3. Extract bacterial DNA from the bacterial culture growing on the Petri dish medium: use the inoculum ring to take out the bacteria in the medium, and then re suspend them at 180 ° by violent stirring μ In lysate 1. Then extract according to the nucleic acid extraction steps.

4. Extract bacterial DNA from liquid culture medium: Take 1ml of 5000g of liquid culture medium and centrifuge for 5 minutes, then resuspend the precipitate at 180 ° C μ Cracking liquid 1. Then extract according to the nucleic acid extraction steps.

5. Extract Gram-positive bacteria DNA: centrifugate 5000g for 10 minutes, then suck out the supernatant, leaving bacterial precipitation; Suspend the bacterial precipitate in a 180 µ l enzyme solution and soak at 37 ° C for at least 30 minutes; Join 25 μ L protease and 200 μ L cracking solution 3, mix well and take a warm bath at 58 ° C for 30 minutes and a warm bath at 95 ° C for 15 minutes; Follow step 5 of nucleic acid extraction to begin extraction.

Note: The enzyme solution can be 20 mg/ml Lysozyme or 200 mg/ml μ G/ml lysostaphin (soluble in 20 mM Tris · HCl, pH 8.0; 2 mM EDTA; 1.2% Triton X-100).

[Operation Steps]

1. Add 25 μ L protease, vortex oscillation for 10 seconds.

2. Place it in a whirlpool shaker and warm it at 58 ℃ until thoroughly cracked (usually 1-3 hours, overnight). If using a dry or water bath that cannot be shaken well, mix well every 15 minutes.

3. 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.

4. Instantaneous centrifugation, adding 200 μ L cracking solution 3, thoroughly mix and soak at 70 ℃ for 10 minutes.

5. Instantaneous centrifugation, adding 200 μ Ethanol (96~100%), cover with lid, vortex oscillate and mix for 15 seconds.

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

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.

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.

10. 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.

11. Bacterial DNA in biological fluid: Centrifuge 5000g for 10 minutes and resuspend the precipitate at 180 ° C μ Cracking liquid 1. Then extract according to the nucleic acid extraction steps.

12. Isolate bacterial DNA from eyes, nose, pharynx or other swabs: collect samples, put them into 2ml PBS containing ordinary Fungicide, and incubate them at room temperature (15-25 ℃) for several hours; Centrifuge 5000g for 10 minutes and resuspend the precipitate at 180 ° C μ Cracking liquid 1. Then extract according to the nucleic acid extraction steps.

13. Extract bacterial DNA from the bacterial culture growing on the Petri dish medium: use the inoculum ring to take out the bacteria in the culture medium, and then suspend them in 180 ° by violent stirring μ In lysate 1. Then extract according to the nucleic acid extraction steps.

14. Extract bacterial DNA from liquid culture medium: Take 1ml of 5000g of liquid culture medium and centrifuge for 5 minutes, then resuspend the precipitate at 180 ° C μ Cracking liquid 1. Then extract according to the nucleic acid extraction steps.

15. Extract Gram-positive bacteria DNA: centrifugate 5000g for 10 minutes, then suck out the supernatant, leaving bacterial precipitation; Suspend the bacterial precipitate in a 180 µ l enzyme solution and soak at 37 ° C for at least 30 minutes; Join 25 μ L protease and 200 μ L cracking solution 3, mix well and take a warm bath at 58 ° C for 30 minutes and a warm bath at 95 ° C for 15 minutes; Follow step 5 of nucleic acid extraction to begin extraction.

Note: The enzyme solution can be 20 mg/ml Lysozyme or 200 mg/ml μ G/ml lysostaphin (soluble in 20 mM Tris · HCl, pH 8.0; 2 mM EDTA; 1.2% Triton X-100)Middle).