Sample Preparation

Plant and animal tissues

20-30 mg of sample weight is recommended. Both fresh and frozen samples can be used.

! Samples can be stored at −70 °С for several months.

1. Place a fresh or a frozen (−70 °С) tissue sample into liquid nitrogen.
2. Transfer the frozen sample to a mortar and thoroughly homogenize it to powder.
3. Transfer the powder into a separate 1.5 mL tube. Wait for liquid nitrogen to evaporate but do not allow the powder to thaw.
4. Add the following reagents to the homogenate in the following order: 300 μL of Lysis Solution NA, 100 μL of water. Mix the contents.
5. Proceed to step 2 of ‘Nucleic Acid Extraction’.

Animal or bacterial cell cultures

Samples of no more than 1-2×10⁶ cell counts for animal cell cultures or 10⁹ cell counts for Gram-negative bacterial cell cultures are recommended.

Adherent cell culture: remove media, harvest cells with trypsin (or with other methods recommended for the cell culture used). Centrifuge the sample at 300 × g for 5 min. Discard the supernatant. Resuspend the cell pellet in 100 μL of PBS. Transfer the suspension into a new 1.5 mL tube.

Animal cells in suspension culture: collect the suspension culture volume required to obtain the desired cell number. Centrifuge cells at 300 × g for 5 min. Discard the supernatant. Resuspend the cell pellet in 100 μL of PBS. Transfer the suspension into a new 1.5 mL tube.

Bacterial cell culture: collect the bacteria grown with liquid or solid media with centrifugation at 3,000-5,000 × g for 5-10 min. Discard the supernatant. Resuspend the cell pellet in 100 μL of PBS. Transfer the suspension into a new 1.5 mL tube.

Proceed to ‘Nucleic Acid Extraction’.

Blood Plasma

This kit is suitable for nucleic acid extraction from blood plasma. Plasma must be collected from peripheral whole blood samples containing EDTA (2.0 mg/mL) or citrate as an anticoagulant.

! Heparin must not be used as an anticoagulant.

! Plasma must be obtained within 6 hours from peripheral blood sample collection.

1. Mix the blood sample by the vial inversion to ensure adequate homogenisation.
2. Centrifuge the vial with the blood sample at 900 × g for 20 min at room temperature (18-25  °С).
3. Aspirate 100 μL of the supernatant (plasma) and transfer to a separate 1.5 mL tube.
4. Proceed to ‘Nucleic Acid Extraction’.

! Store the plasma sample at −20 °С for no more than 3 months.

Epithelial cells in swab samples

This kit is suitable for total nucleic acid extraction from swab samples of epithelial cells collected with a single-use sterile swab (buccal, posterior pharynx, nasopharyngeal, urethral, cervical, vaginal swabs, etc.).

1. Place 500 μL of sterile saline into a 1.5 mL tube.
2. Vigorously swirl the swab to resuspend the sample material in saline. Press the swab against the wall of the vial and squeeze out the residual saline with a circular motion.
3. Centrifuge the solution at 11,000 × g for 10 min. Thoroughly remove the supernatant.
4. Resuspend the pellet in 100 μL of sterile saline.
5. Proceed to ‘Nucleic Acid Extraction’.

Urine

1. Transfer 1 mL of a urine sample into a clean 1.5 mL tube.
2. Centrifuge at 11,000 × g for 10 min. Thoroughly remove the supernatant.
3. Resuspend the pellet in 1 mL of sterile saline.
4. Centrifuge at 11,000 × g for 10 min. Discard the supernatant.
5. Resuspend the pellet in 100 μL of sterile saline.
6. Proceed to ‘Nucleic Acid Extraction’.

Saliva, liquor, synovial fluid

1. Add 500 μL of the sample into a separate 1.5 mL tube.
2. Centrifuge at 11,000 × g for 10 min. Thoroughly remove the supernatant leaving ~50 μL of the solution above the pellet.
3. Resuspend the pellet in 500 μL of sterile saline.
4. Centrifuge at 11,000 × g for 10 min. Discard the supernatant.
5. Resuspend the pellet in 100 μL of sterile saline.
6. Proceed to ‘Nucleic Acid Extraction’.

Semen, prostate secretes

1. Add 100 μL of the sample into a separate 1.5 mL tube.
2. Add 500 μL of sterile saline into the tube, vortex for 5-10 sec.
3. Centrifuge at 11,000 × g 10 min. Discard the supernatant.
4. Resuspend the pellet in 100 μL of sterile saline.
5. Proceed to ‘Nucleic Acid Extraction’.

Smears and washing fluids

1. Place the specimen to the centrifugation vial.
2. Centrifuge at 11,000 × g 10 min. Discard the supernatant.
3. Resuspend the pellet in 100 μL of sterile saline.
4. Proceed to ‘Nucleic Acid Extraction’.

Feces

1. Transfer 1 mL of sterile saline into a clean 1.5 mL tube.
2. Add ~250 mg (μL) of feces into the tube.
3. Vortex the content for 5-10 sec.
4. Centrifuge at 100 × g for 3 min.
5. Transfer 800-1,000 μL of supernatant into a separate 1.5 mL tube.
6. Centrifuge for at 11,000 × g 10 min. Discard the supernatant.
7. Resuspend the pellet in 100 μL of sterile saline.
8. Proceed to ‘Nucleic Acid Extraction’.

Sputum specimen

1. Add mucolysin to a vial containing the sample in the ratio of 5:1 (5 parts mucolysin and 1 part of sputum) using graduated marks on the vial.
2. Close the vial lid, shake the content. Incubate for 20-30 min at room temperature, shake the vial every 2-3 min.
   ! The processed sputum sample can be stored at 4 °С for 24 hours or at −20 °С for long-term.
3. Transfer 500 μL of the mucolysin-treated sputum sample into a separate 1.5 mL tube.
4. Centrifuge for at 11,000 × g 10 min. Discard the supernatant.
5. Resuspend the pellet in 100 μL of sterile saline.
6. Proceed to ‘Nucleic Acid Extraction’.

Nucleic Acid Extraction

All the procedures should be performed at room temperature; >13,000 rpm (>11,000 × g) centrifugation should be used unless directed otherwise.

Before you begin, set the dry block heater to 65 °С and preheat the vial with Resuspension Buffer NA.

Prepare the specimen in 100 μL volume in a 1.5 mL tube as directed in ‘Sample Preparation’.

1. Add 300 μL of Lysis Solution NA to the vial containing the sample (100 μL), and mix thoroughly by vortexing.
2. Incubate the resulting solution at 65 °С for 15 min.
3. (Optional) If the sample is not fully dissolved after lysis, centrifuge the vial for 10 min. Transfer the supernatant into a new 1.5 mL tube.
4. Add 400 μL of Precipitation Solution to the vial containing the sample and vortex. Centrifuge for 15 min.
5. Carefully remove the supernatant, take care to avoid disturbing the pellet. Add 500 μL of Washing Solution 1 to the pellet and mix by vortexing. Centrifuge for 5 min.
6. Carefully remove the supernatant, take care to avoid disturbing the pellet. Add 500 μL of Washing Solution 2 to the pellet and mix by vortexing. Centrifuge for 5 min.
7. Thoroughly remove the supernatant, avoid disturbing the pellet. Open the vial and dry the pellet at 65 °С for 5 min.
8. Add 50 μL of preheated Resuspension Buffer NA.
9. Incubate the vial containing the specimen at 65 °С for 5-10 min. Mix the content by vortexing and centrifuge to collect drops. The resulting product of total nucleic acid is suitable for downstream PCR or RT-PCR without additional processing.

Extracted RNA Storage: We do not recommend storing extracted RNA due to its instability. The extracted RNA product must be used in the downstream reverse transcription-polymerase chain reaction immediately.

Extracted DNA Storage: Store at 4 °С for short-term; store at −20 °С for no more than 1 month or at −70 °С for no longer than 1 year.

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