DNA Extraction Kit Column

Lysis occurs by thermal shock, and the purification by a series of processes to remove contaminants and residual cellular debris. The clear supernatant is then mixed with the Binding buffer, to create conditions for optimal binding to the silica membrane column. The purified DNA is ready to use for subsequent reactions like real-time PCR detection and quantification.

Applications

• DNA from complex matrices, processed food, raw food and animal feed, soya, chocolate, cereals, meat
• Detection of specific DNA in animal feed
• Detection of genetically modified material in food products (GMO)
• Extraction of microbial DNA
• DNA extraction from swabs and sponges
• DNA suitable for PCR, real-time PCR, enzymatic reactions

If properly stored, see the expiration date for the stability of the kit. RT: room temperature.

(1) Reconstitute Proteinase K by adding nuclease-free water (Molecular Biology grade) as indicated on the vial(s) and store at -20 °C. It is recommended to make several aliquots to avoid thaw/freeze cycles. At this temperature it is stable for 1 year.

(2) Add ethanol (96-100%) to the Wash buffers prior to use, as indicated on the bottle(s). Keep the containers closed to avoid the ethanol evaporation.

• Microcentrifuge tubes (1.5-2.0 mL; 15-50 mL)
• Micropipettes and micropipette filter tips (10-100 µL and 100-1000 µL)
• Vortex
• Microcentrifuge, able to operate up to 10,000 g
• Heater block (preferably) or water bath at 37 °C, 65 ± 5 °C to 95 ± 5 °C
• Powder-free gloves
• Ethanol 100%
• Real-time detection kits for allergens or pathogens (optional)

• These products are exclusively for in vitro use.
• 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.
• The Lysis buffer, Binding buffer and Wash buffer 1 contain guanidine hydrochloride, which can form reactive compounds when combined with bleach (sodium hypochlorite).
• 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 the Lysis buffer. 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.

Preliminary preparations

Make sure that Proteinase K and the Wash buffers were prepared according to the Contents and storage section.

Preheat the Lysis buffer and Elution buffer to 65-70 °C.

Procedure 1: DNA isolation from food

(For dairy products see procedure 2.)

Sample homogenization:

1. Weigh about 200 mg of food material and transfer it to a microcentrifuge tube.

To obtain a good yield, good homogenization of the sample is necessary. The lysis procedure is most effective when well-homogenized samples are used. We recommend grinding with a pestle and mortar or commercial homogenizers.

In liquid samples use 200 µL directly.

For dairy products see procedure 2.

Cell lysis step:

2. Pipette 1000 µL of Lysis buffer (preheated) into the microcentrifuge tube, and add 25 µL of Proteinase K. Vortex vigorously.
3. Apply a short spin down and incubate for 30 minutes at 65 °C. Vortex once or twice during incubation (optional).

For samples with high fat content (e.g. chorizo, cheese, ham, etc.) an O/N lysis step (16-18 h at 65 °C) and double the amount of Proteinase K (50 µL) may be necessary.

4. Centrifuge at > 10,000 g for 5 minutes.

DNA binding step:

5. Transfer 500 µL of clear supernatant to a new microtube with 250 µL of Binding buffer. Vortex briefly.

On the surface, a layer of fat could appear. To collect a clear supernatant, introduce the pipette tip crossing this superficial layer of fat, only trying to pick up the supernatant liquid with color (and avoid touching the pellet with the tip).

6. Place the spin column in a 2 mL collection tube.
7. Transfer 600 µL of the mix to the column, and centrifuge at > 10,000 g for 1 minute. Discard the flow-through.
8. Repeat step 7 to load the remaining sample.
9. Place the spin column in a new collection tube.

Washing step:

10. Add 500 µL of Wash buffer 1 and centrifuge at > 10,000 g for 1 minute. Discard the flow-through.
11. Add 700 µL of Wash buffer 2 and centrifuge at > 10,000 g for 1 minute. Discard the flow-through.
12. Centrifuge at > 10,000 g for 3 minutes.

Elution step:

13. Transfer the spin column to a new 1.5 mL microcentrifuge tube and pipette 100 µL of Elution buffer (preheated at 70 °C) onto the membrane. Incubate at room temperature for 2 minutes.

Decrease the volume of Elution buffer (such as 50 µL) if a higher DNA concentration is desired.

14. Centrifuge at > 10,000 g for 1 minute. Discard the spin column and use the DNA immediately or store it at -20 °C.

Procedure 2: DNA isolation from dairy products

Before starting, preheat the Lysis buffer and Elution buffer to 70 °C.

Sample homogenization:

1. Homogenize the food sample and centrifuge about 50 mL at > 10,000 g for 10 minutes. Discard the supernatant.

To obtain a good yield, good homogenization of the sample is necessary. The lysis procedure is most effective when well-homogenized samples are used. We recommend this procedure for samples of dairy products such as milk, butter and cream cheese or cottage cheese. For other types of cheese, weigh 300 mg of food material and then go directly to step 4 of this procedure.

2. Centrifuge for another 10 minutes at > 10,000 g. Discard the supernatant.
3. Wash the pellet with 1 mL of nuclease-free water or PBS. Then centrifuge for 3 minutes at > 10,000 g.
4. Pipette 1000 µL of Lysis buffer (preheated) into the microcentrifuge tube, and add 50 µL of Proteinase K. Vortex vigorously.
5. Apply a short spin down and incubate for 30 minutes at 65 °C. Vortex once or twice during incubation.

OPTIONAL: O/N lysis step (16-18 h at 65 °C).

6. Centrifuge at > 10,000 g for 5 minutes.
7. Proceed to the DNA binding, washing and elution steps of procedure 1, DNA isolation from food.

Procedure 3: bacterial DNA isolation from enrichment culture

Before starting, preheat the Lysis buffer and Elution buffer to 70 °C.

Concentration step:

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.

Cell lysis step:

2. Pipette 500 µL of Lysis buffer (preheated) into the microcentrifuge tube. Vortex vigorously.
3. Apply a short spin down and incubate for 15 minutes at 95 °C.
4. Centrifuge at > 10,000 g for 5 minutes.
5. Proceed to the DNA binding, washing and elution steps of procedure 1, DNA isolation from food.

Procedure 4: DNA isolation from swabs and sponges

Before starting, preheat the Lysis buffer and Elution buffer to 70 °C.

Cell lysis step:

1. Add the swab/sponge to 1000 µL of Lysis buffer (preheated) in the microcentrifuge tube, and add 25 µL of Proteinase K. Vortex vigorously.
2. Incubate at room temperature for 2 hours with shaking.
3. Take out the swab/sponge by pressing it against the wall of the microcentrifuge tube in order to recover all the sample.
4. Apply a short spin down and incubate for 30 minutes at 65 °C. Vortex once or twice during incubation (optional).
5. Centrifuge at > 10,000 g for 5 minutes.
6. Proceed to the DNA binding, washing and elution steps of procedure 1, DNA isolation from food.

Procedures 1 and 3: food and enrichment culture

1. Food homogenization: homogenize and weigh 200 mg of food (for other procedures see the complete protocol).
2. Lyse cells: 1000 µL Lysis buffer + 25 µL Proteinase K; 65 °C, 30 min (food) or 500 µL Lysis buffer, 95 °C, 15 min (bacteria); > 10,000 x g, 5 min; take 500 µL of supernatant and continue with step 4.
3. Bind DNA: 500 µL of supernatant (step 3); 250 µL Binding buffer; load the spin column (max. 600 µL); > 10,000 x g, 1 min.
4. Wash: 500 µL of Wash buffer 1, > 10,000 x g, 1 min; 700 µL of Wash buffer 2, > 10,000 x g, 1 min; > 10,000 x g, 3 min.
5. Elute DNA: 100 µL of Elution buffer (preheated at 70 °C); > 10,000 x g, 1 min.

Procedure 2: dairy food

1. Food homogenization: homogenize and weigh 50 mL of food (for each type of food see the complete protocol).
2. Wash pellet: > 10,000 x g, 10 min; > 10,000 x g, 10 min; 1 mL nuclease-free water (or PBS); > 10,000 x g, 3 min.
3. Lyse cells: 1000 µL Lysis buffer + 50 µL Proteinase K, 65 °C, 30 min (food); > 10,000 x g, 5 min; take 500 µL of supernatant and continue with step 4.
4. Bind DNA: 500 µL of supernatant (step 3); 250 µL Binding buffer; load the spin column (max. 600 µL); > 10,000 x g, 1 min.
5. Wash: 500 µL of Wash buffer 1, > 10,000 x g, 1 min; 700 µL of Wash buffer 2, > 10,000 x g, 1 min; > 10,000 x g, 3 min.
6. Elute DNA: 100 µL of Elution buffer (preheated at 70 °C); > 10,000 x g, 1 min.

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.