DNA Extraction Kit Rapid

Lysis occurs by thermal shock and a centrifugation step removes part of the residual cellular debris. The supernatant could be used directly for subsequent reactions like real-time PCR detection, or after a dilution step to remove residual PCR inhibitors.

Store at room temperature (RT).

• Microcentrifuge tubes (1.5-2.0 mL)
• Micropipettes and micropipette filter tips (10-100 µL and 100-1000 µL)
• Vortex
• Microcentrifuge, able to operate up to 14,000 g
• Heater block (preferably) or water bath at 95 ± 5 °C
• Powder-free gloves

• These products are exclusively for in vitro use, for Research Use Only (RUO).
• These products are for food use only.
• The test requires qualified staff to prevent the risk of erroneous results.
• Do not mix reagents from different batches.
• Do not use reagents from other manufacturers' products.
• Wear disposable gloves and laboratory coats when handling specimens and reagents.
• Use sterile pipette tips with filters.
• Avoid contact of specimens and reagents with the skin, eyes and mucous membranes. If these solutions come into contact, rinse immediately with water and seek medical advice immediately.
• Material Safety Data Sheets (MSDS) are available on request.
• Waste must be treated and disposed of in compliance with the appropriate safety standards.
• Clean the working space periodically with at least 5% sodium hypochlorite.
• It is strongly recommended to have dedicated areas, materials, and equipment for the DNA extraction, preparation of the PCR and post-PCR procedures.

The kit contents should be mixed slightly before use.

Under cool environmental conditions, a precipitate may form in some kit components. In this case, the component should be heated for approximately 5 minutes at 37 °C to dissolve the precipitate and thoroughly shaken prior to use.

Procedure 1: bacterial DNA isolation from pre-enrichment or enrichment culture

Sample concentration:

1. Centrifuge 1 mL of pre-enrichment or enrichment medium at > 10,000 g for 5 minutes. Discard the supernatant.

Avoid transferring food debris from the enrichment medium into the microcentrifuge tube.

OPTIONAL: Perform a washing step before step 2: add 1 mL of sterile PBS, resuspend the pellet and centrifuge at > 10,000 g for 3 minutes. Discard the supernatant. Continue to step 2.

Cell lysis step:

2. Pipette 100-200 µL of Lysis buffer into the microcentrifuge tube and resuspend the pellet. Vortex vigorously.
3. Apply a short spin down and incubate for 10-15 minutes at 95-100 °C.
4. Centrifuge at > 10,000 g for 5 minutes.
5. Transfer the clear supernatant to a new microcentrifuge tube.
6. Use the DNA immediately or store it at -20 °C if you want to reuse it. Homogenize and centrifuge at > 10,000 g for 2 minutes before reusing.

Procedure 2: DNA isolation from dairy products

1. Pipette 100 µL of Lysis buffer into the microcentrifuge tube.
2. Select a small loopful of cells on a culture plate and suspend the cells in the Lysis buffer reagent. Vortex vigorously.
3. Apply a short spin down and incubate for 10-15 minutes at 95-100 °C.
4. Centrifuge at > 10,000 g for 5 minutes.
5. Transfer the clear supernatant to a new microcentrifuge tube.
6. Use the DNA immediately or store it at -20 °C if you want to reuse it. Homogenize and centrifuge at > 10,000 g for 2 minutes before reusing.

Procedure 3: bacterial DNA isolation from enrichment culture

Concentration step:

1. Centrifuge up to 50 mL at maximum speed for 10 minutes. Decant the liquid carefully, leaving about 1 mL in the cone of the tube.

(Centrifuge volumes between 1 mL and 50 mL.)

Wash step:

2. Mix the liquid in the cone with a pipette tip, transfer it completely into a 1.5 mL microtube and centrifuge for 3 minutes at > 10,000 g.
3. With a pipette tip, remove all the supernatant.
4. Pipette 700 µL of sterile PBS and resuspend the pellet by vortexing or pipetting up and down. Centrifuge for 3 minutes at > 10,000 g.
5. With a pipette tip, remove all the supernatant. Repeat step 4 one more time (wash buffer and centrifugation).
6. With a pipette tip, remove all the supernatant.

Cell lysis step:

7. Pipette 100 µL of Lysis buffer into the microcentrifuge tube and resuspend the pellet by vortexing or pipetting up and down. (If the pellet is not completely covered, increase the volume of Lysis buffer, no more than 200 µL.)
8. Add 3 µL of Lyticase (15 U). (Add 3 µL of lyticase for each 100 µL of Lysis buffer added in step 7.)
9. Vortex and incubate for 15 minutes at 37 °C.
10. Incubate for 10-15 minutes at 95-100 °C.
11. Centrifuge at > 10,000 g for 5 minutes.
12. Transfer the clear supernatant to a new microcentrifuge tube.
13. Use the DNA immediately or store it at -20 °C if you want to reuse it. Homogenize and centrifuge at > 10,000 g for 2 minutes before reusing.

Procedure 1: enrichment culture

1. Cell concentration: collect 1 mL of medium; > 10,000 x g, 5 min.
2. Lyse cells: 100-200 µL Lysis buffer; 95/100 °C, 10-15 min; > 10,000 x g, 5 min; take the supernatant.

Procedure 2: isolated colonies

2. Lyse cells: select a small loopful of cells; 100 µL Lysis buffer; 95/100 °C, 10-15 min; > 10,000 x g, 5 min; take the supernatant.

Procedure 3: DNA from beverages

1. Cell homogenization: collect 1-50 mL; max speed, 10 min; decant the supernatant.
2. Wash cells: > 10,000 x g, 3 min; remove the supernatant; 700 µL PBS; > 10,000 x g, 3 min; remove the supernatant; repeat: 700 µL PBS; > 10,000 x g, 3 min; remove the supernatant.
3. Lyse cells: 100-200 µL Lysis buffer; 3-6 µL Lyticase; 37 °C, 15 min; 95-100 °C, 10-15 min; > 10,000 x g, 5 min; use DNA directly or diluted.

Each lot of the kit is tested against predetermined specifications to ensure consistent product quality.

The kit must be used solely in accordance with the respective Instructions for Use.

The kit components are intended exclusively for in vitro use, and for research purposes only. The products are intended for general laboratory use only.

Molecular biology procedures, such as DNA extractions and PCR amplification, require qualified staff to prevent the risk of erroneous results. It is strongly recommended to have dedicated areas, materials and equipment for the DNA extraction, preparation of the PCR and post-PCR procedures. The workflow in the laboratory should proceed in a unidirectional manner, from the Extraction Area to the Amplification and Detection Area. Do not return samples, equipment and reagents to the area where you performed the previous step. The user should always read all the instructions provided with the product before running the assay. Do not mix reagents from different batches. Do not use reagents from other manufacturers' products. Wear disposable gloves and laboratory coats when handling specimens and reagents. Use sterile pipette tips with filters. Waste must be treated and disposed of in compliance with the appropriate safety standards.