Bacterial genomic DNA extraction reagent instruction manual

Bacterial genomic DNA extraction reagent instruction manual

â—† Product Description

Based on silica gel column purification. Lysozyme digestion in the reagent removes the bacterial cell wall. Gram-positive bacteria can also be vortexed by glass beads, lysed by lysate and protease, and released into the lysate. After adding ethanol, it is transferred to an adsorption column for filtration, and the DNA is adsorbed onto the membrane of the adsorption column, and the protein is removed. The adsorption column is washed by Buffer GW2 to remove other impurities, and finally the DNA is eluted by low salt buffer (Buffer AE, etc.), and the eluted DNA can be directly used for constant temperature fluorescence detection and real-time PCR detection.

â—† Product composition

• Storage conditions and expiration date

Proteinase K dry powder and Lysozyme are dry powder, transported at room temperature, stored at 2 - 8 ° C or -20 ° C, and other components can be stored at room temperature with a shelf life of one year. At low temperatures, Buffer SDS or a small amount of precipitate formed and dissolved at 37 ° C.

• Self-contained materials

• Anhydrous ethanol (≥96%);
• Centrifuge tube and tip;
• Centrifuge
• Water bath or metal bath

â—† Reagent preparation

1) Add 0.6 ml of Protease Dissolve Buffer to the Proteinase K dry powder, mix by inversion to dissolve the proteinase K, and store at -20 °C.

2) Add 2 ml of Protease Dissolve Buffer to the Lysozyme tube, mix by inversion to dissolve the lysozyme and store at -20 °C.

3) Dilute Buffer GW2 with 40 ml of absolute ethanol and store at room temperature.

â—†How to use

I. Gram-negative bacterial DNA extraction

This protocol is suitable for extracting total DNA from <1.5 × 10 ⁹ Gram-negative bacteria. The amount of bacteria can be measured by a spectrophotometer. The 1OD ₆₀₀ is approximately 1.5 x 10 ⁹ Gram-negative bacteria.

• Transfer 0.5-1 ml of bacterial culture (<1.5 × 10 ⁹ bacteria) to a 1.5 ml centrifuge tube. The bacteria were collected by centrifugation at 10,000 rpm for 1 minute, and the culture solution was discarded.
• 200 μl Buffer STE, 10 μl Lysozyme and 10 μl Proteinase K were added to the bacterial pellet. The bacteria were fully resuspended in a vortex and incubated at 37 ° C for 10 to 30 minutes.
• Add 220 μl Buffer DL to the bacterial resuspension. The mixture was vortexed and digested in a 65 ° C water bath for 30 minutes.
• 220 μl of absolute ethanol was added to the lysate. The highest speed vortex for 30 seconds.

Note: If there is a flocculent precipitate in this step, use a pipette to try to break up the sediment as much as possible.

• The adsorption column Column was placed in a 2 ml collection tube. Transfer the mixture obtained in step 4 (including precipitation) to the adsorption column. Centrifuge at 10,000 rpm for 1 minute.

Note: If the adsorption column is clogged, increase the centrifugal speed to 13,000 rpm and centrifuge for 3 minutes.

• The filtrate was discarded and the column was returned to the collection tube. 600 μl of Buffer GW2 (diluted with ethanol) was added to the adsorption column. Centrifuge at 10,000 rpm for 1 minute.

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

• The filtrate was discarded and the column was returned to the collection tube. An additional 300 μl of Buffer GW2 (diluted with ethanol) was added to the adsorption column. Centrifuge at 10,000 rpm for 3 minutes.
• The column was packed in a new 1.5 ml centrifuge tube. Add 30-50 μl of preheated to 65 ° C Buffer AE to the center of the adsorption column membrane. Leave for 3 minutes. Centrifuge at 10,000 rpm for 1 minute.

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

DNA extraction of Gram-positive bacteria

This protocol is suitable for extracting total DNA from <1.5 × 10 ⁹ Gram-positive bacteria. The amount of bacteria can be measured by a spectrophotometer. 1 OD ₆₀₀ is approximately 1.5 × 10 ⁹ Gram-positive bacteria. The cell wall of Gram-positive cells is very thick and cannot be directly lysed. The cell wall is removed by lysozyme digestion. Gram-negative bacteria and most Gram-positive bacteria such as bacilli can be well treated by enzymatic treatment to achieve good wall breaking effect. Certain Gram-positive bacteria have a thick bacterial wall and must be combined with bead milling to achieve results.

• Enzymatic method: The cell wall is digested with Lysozyme to convert the bacteria into protoplasts. The genus Staphylococcus contains thicker cell walls and is also required to be digested together with Lysostaphin.
• Bead-milling enzyme method: The cell wall is first digested with lysozyme, and then bacteria are lysed by high-speed vortexing with pickled glass beads (0.1-0.2 mm). When treating bacteria such as staphylococcus and faecal bacteria, the addition of glass beads vortex can significantly increase the yield.

• Transfer 0.5-1 ml of bacterial culture (<1.5 × 10 ⁹ bacteria) to a 1.5 ml centrifuge tube. The bacteria were collected by centrifugation at 10,000 rpm for 1 minute, and the culture solution was discarded.

Note: If the culture medium density is relatively large, the centrifugation speed and centrifugation time may need to be increased to ensure sufficient sediment collection of bacteria. If you need to extract DNA from the plaque: use the inoculation ring to scrape the plaque, follow step 2, transfer the plaque to Buffer STE, and vortex to mix.

• Add 200 μl Buffer STE and 30 μl Lysozyme to the bacterial pellet. The bacteria were fully resuspended in a vortex and bathed at 37 ° C for 30 minutes. When treating bacteria of the genus Staphylococcus, it is preferable to add 1 μl of Lysostaphin (20 mg/ml).
• Add 15 μl Buffer SDS and 10 μl Proteinase K to the bacterial suspension. The mixture was vortexed and digested in a 65 ° C water bath for 30 minutes.
• Centrifuge at 10,000 rpm for 3 minutes at room temperature. Transfer the supernatant to a new centrifuge tube.
• 250 μl of Buffer DL and 250 μl of absolute ethanol were added to the lysate. The highest speed vortex for 30 seconds.

Note: If there is obvious flocculent precipitation in this step, use a pipette to suck as much as possible to break up the sediment.

• The adsorption column Column was placed in a 2 ml collection tube. Transfer the mixture obtained in step 5 (including precipitation) to the adsorption column. Centrifuge at 10,000 rpm for 1 minute.

Note: If the adsorption column is clogged, increase the centrifugal speed to 13,000 rpm and centrifuge for 3 minutes.

• The filtrate was discarded and the column was returned to the collection tube. Add 600 μl Buffer GW2 (diluted with ethanol) to the adsorption column. Centrifuge at 10,000 rpm for 1 minute.

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

• The filtrate was discarded and the column was returned to the collection tube. An additional 300 μl of Buffer GW2 (diluted with ethanol) was added to the adsorption column. Centrifuge at 10,000 rpm for 2 minutes.
• The column was packed in a new 1.5 ml centrifuge tube. Add 30-50μl to preheat to 65oC Buffer AE to the center of the adsorption column membrane. Leave for 3 minutes. Centrifuge at 10,000 rpm for 1 minute.
• Discard the DNA binding column, store the DNA at 2-8 ° C, and store it at -20 ° C for long-term storage.

3. Extraction of bacterial DNA from tissue or liquid samples

The program is suitable for extracting genomic DNA and parasitic bacterial DNA from liquid samples such as various tissues, blood, and secretions. The purified DNA can be directly used for the detection of various bacteria. To extract bacterial DNA from stool samples, we recommend the DePure Stool DNA Kit. If you need to extract bacterial DNA from soil samples, the DePure Soil DNA Kit is recommended.

• Pre-process as follows:

• Solid sample: 10 mg of the solid sample was cut into small pieces and transferred to a 1.5 ml centrifuge tube, and 200 μl of Buffer STE and 10 μl of Buffer SDS were added.
• Whole blood samples: Cells and bacteria were isolated by routine procedure using <1 ml of anticoagulant. Add 200 μl Buffer STE and 10 μl Buffer SDS and resuspend the cells with a pipette.
• Secretion or serum, etc.: Centrifuge at 10,000 × g for 3 minutes to collect cells and bacteria; discard the supernatant, add 200 μl Buffer STE and 10 μl Buffer SDS, and resuspend the cells with a pipette.
• Viscous secretion: Take 100 μl or 100 mg of sputum, etc., add 100 μl of Buffer STE, 10 μl of Buffer SDS and 5 μl of 1 M dithiothreitol DTT. (If you need to isolate Mycobacterium tuberculosis or fungi from sputum, you can first liquefy the sputum with NaOH, centrifuge to collect the precipitate and then operate.)

• Add 10 μl of Proteinase K to the resuspension. Vortex and mix. Leave the water bath at 55 ° C for 1-3 hours or until the sample is completely digested. Operate according to the extraction process of bacteria and cell total DNA, or the difficult-to-crack bacterial extraction process.

Bacterial and cell total DNA extraction

• (Optional) Further lyse the bacteria in a 95 ° C water bath for 10 minutes.
• Centrifuge at 10,000 rpm for 3 minutes. Transfer the supernatant to a new 1.5 ml centrifuge tube.
• 200 μl of Buffer DL and 200 μl of absolute ethanol were added to the supernatant. Vortex for 20 seconds.
• Follow steps 7-12.

Difficult to lyse bacterial DNA extraction

• Undigested bacteria were collected by centrifugation at 10,000 rpm for 3 minutes. Aspirate all supernatant.
• Add 200 μl Buffer STE and 30 μl Lysozyme to the bacterial pellet. The bacteria were fully resuspended in a vortex and incubated at 37 ° C for 30-60 minutes. When treating bacteria of the genus Staphylococcus, it is preferable to add 1 μl of Lysostaphin (20 mg/ml).
• Add 20 μl Buffer SDS to the bacterial resuspension. Mix by vortexing and water bath at 95 ° C for 10 minutes.
• 250 μl of Buffer DL and 250 μl of absolute ethanol were added to the lysate. The highest speed vortex for 30 seconds. Follow steps 7-12.

Adsorption of DNA through the column

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

Note: If the adsorption column is clogged, increase the centrifugal speed to 13,000 rpm and centrifuge for 3 minutes.

• The filtrate was discarded and the column was reloaded back into the collection tube. Add 650 μl Buffer GW2 (diluted with ethanol) to the adsorption 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 300 μl Buffer GW2 (diluted with ethanol) to the adsorption column. Centrifuge at 10,000 rpm for 2 minutes.
• The column was packed in a new 1.5 ml centrifuge tube. Add 30-100 μl of preheated to 65 ° C Buffer AE to the center of the adsorption column membrane. Leave for 3 minutes. Centrifuge at 10,000 rpm for 1 minute.
• Add 30-100 μl of preheated to 65 ° C Buffer AE to the center of the membrane of the adsorption column. Leave for 3 minutes. Centrifuge at 10,000 rpm for 1 minute.

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

4. Total bacterial DNA extraction from swab samples

This protocol is suitable for extracting genomic DNA and total bacterial DNA from various swab samples.

• Transfer the swab sample to a 2 ml centrifuge tube. 350 μl Buffer STE and 30 μl Lysozyme were added to the swab samples. The bacteria were fully resuspended in a vortex and incubated at 37 ° C for 10-30 minutes. When treating bacteria of the genus Staphylococcus, it is preferable to add 1 μl of Lysostaphin (20 mg/ml).
• Add 30 μl Buffer SDS and 10 μl Proteinase K to the bacterial resuspension. The mixture was vortexed and digested in a 65 ° C water bath for 30 minutes.
• (Optional) Further lysing the lysable bacteria by a 95 ° C water bath for 10 minutes.
• 400 μl of Buffer DL and 400 μl of absolute ethanol were added to the lysate. Vortex for 20 seconds.
• Install the DNA column in the collection tube. 600 μl of the mixed solution (the mixture obtained in the fourth step) was transferred to the adsorption column. Centrifuge at 10,000 rpm for 1 minute.
• The effluent is discarded and the column is returned to the collection tube. Transfer the remaining mixture to the adsorption column. Centrifuge at 10,000 rpm for 1 minute.
• The filtrate was discarded and the column was reloaded back into the collection tube. Add 650 μl Buffer GW2 (diluted with ethanol) to the adsorption 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 300 μl Buffer GW2 (diluted with ethanol) to the adsorption column. Centrifuge at 10,000 rpm for 2 minutes.

9. Place the column in a new 1.5 ml centrifuge tube. Add 30-50 μl of preheated to 65 ° C Buffer AE to the center of the adsorption column membrane. Leave for 3 minutes. Centrifuge at 10,000 rpm for 1 minute. Discard the DNA binding column, store the DNA at 2-8 ° C, and store it at -20 ° C for long-term storage.

【 Notes 】

1. Work clothes, latex gloves and masks were worn during the experiment, and pipettes were used to process all the waste generated in the test in time.

2. Please strictly follow the operation steps. If you have any technical problems, please contact us in time.

3. Please use the kit during the validity period.

YT-T13

YT-T13

YT-T13

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