DNA Extraction Kit Complex

The samples contained in a microtube are subjected to alkaline lysis in the absence of chaotropic agents but with a combination of resins that retain different organic molecules and ions, which could affect the quality of the DNA.

In a last step, the sample is resuspended with a pH correcting buffer containing neutralizing agents for large PCR inhibitors (for example, humic acids) not captured by the resins.

Table 1. Sample examples to process.

Contents

This product contains 2 reagents, enough to extract DNA from 100 samples.

• R1: Lysis buffer, 25 mL
• R2: Inhibitor Neutralization and Adjustment Buffer, 75 mL

Additional equipment required

• Variable volume pipette 100-1000 µL.
• Filter pipette tips.
• Thermal block with agitation, 100-95 °C.
• Boil-proof microcentrifuge tubes.
• Tube rack.
• Microcentrifuge.
• Vortex or tube mixers (1000 rpm, at least).

Storage and preservation

This kit is sent at room temperature; it can be kept without refrigeration until the moment of opening. Once opened, keep refrigerated (2-8 °C); it will remain stable for up to one year.

Microbiological state

Sterile products.

Preparation of reagents

This reagent is ready for use.

General rules

Carefully read the safety data sheets for this and all the products that you will use throughout the trial. The reagents R1 and R2 have extreme pH; do not handle them without first checking the safety data sheet.

This product is manufactured and sold for quality control in food/environmental samples; it has not been designed for any other use, especially for the diagnosis of infectious diseases. Its use must be carried out by personnel experienced and qualified in the handling of potentially dangerous chemical products.

Users must make independent decisions about the integrity of the information based on all available sources.

Actions to be performed before starting

All PCR workflows must be carried out under conditions that prevent environmental contamination. It is recommended that in each series you include at least one blank to keep contamination risks under control.

• Remove reagents R1 and R2 from the refrigerator.
• Start the thermal block, and set the temperature to 80-90 °C.
• Put R1 on the magnetic stirrer, at a sufficient speed to guarantee a correct homogenization.

The protocol begins on a pellet of the sample, obtained by centrifuging at least at 10-13,000 g for 5-10 minutes, which can have a diverse nature (see Table 1).

Protocol

1. Add 0.2 mL of R1.
2. Shake at maximum speed (minimum 1000 rpm) for 5 min.
3. Incubate at 80-90 °C while shaking for 10 minutes. If a stirred thermal block is not available, homogenize 2-3 times, with the help of a vortex, during the heat treatment.
4. Remove the tubes and dispense 0.6 mL of R2.
5. Shake at maximum speed (minimum 1000 rpm) for 1-2 min.
6. Centrifuge at 10-13,000 g for 1-2 minutes.

The supernatant can be used immediately in a PCR. If it is necessary to conserve the DNA, transfer 0.3 mL to a new tube and store at least at -20 °C.

Note: If you are going to measure the quantity of DNA and its purity by spectrophotometric methods, remember that the equipment blank cannot be performed with water or TE buffer; it must be performed with a solution based on the reagents used (1 vol. of R1 + 3 vol. of R2), otherwise you will be overestimating the amount of DNA and its impurity.

The use of this product is not intended for therapeutic purposes or domestic, agricultural or cosmetic use. It must be used under the supervision of a technically qualified person experienced in the handling of potentially hazardous chemical products.