Sample Preparation for DNA and RNA Extraction:
A Practical Pretreatment Guide
Find your sample type, then choose the extraction target. The tables give representative sample amounts, reagent volumes, centrifugation conditions and essential digestion or incubation steps. Routine methods appear first, with special treatments beneath them.
Choose the fraction before choosing the treatment. Cellular DNA or RNA, cell-free nucleic acids and pathogen nucleic acids may require different preparations from the same sample. Check whether the target is in the pellet, the liquid or both. PBS and saline disperse samples; they do not replace a lysis reagent.
Using the examples: Quantities belong to the named Magen protocol and its prepared reagents. For an unlisted sample, match both the matrix and the target, confirm compatibility with the selected kit and test a small input first. These examples include essential lysis, digestion and clarification, but omit routine nucleic acid binding, washing and elution. Follow the kit for subsequent purification. Centrifugation is expressed as relative centrifugal force (× g); rpm is used only for shaking or grinding. RT means room temperature.
Quick Index by Sample Type
- 1. Blood: Whole blood and buffy coat; direct lysis or red blood cell removal according to the extraction target.
- 2. Plasma and Serum: Cell-free DNA, cell-free RNA including miRNA, viral nucleic acids and extracellular-vesicle-associated nucleic acids.
- 3. Animal Tissues: Routine animal tissues and insect tissues; preparation for DNA, RNA, high-molecular-weight DNA or pathogen nucleic acids.
- 4. FFPE Tissues: Formalin-fixed, paraffin-embedded tissue sections; DNA, RNA or separate recovery of both from one sample.
- 5. Cultured Cells: Suspension and adherent cells; cell collection or direct lysis.
- 6. Other Clinical Samples: Urine, amniotic fluid, saliva, swabs, sputum, bronchoalveolar lavage fluid, cerebrospinal fluid and ascites.
- 7. Plant Tissues: Fresh, frozen or dried plant tissues; routine, polyphenol-rich and other difficult plant matrices.
- 8. Bacterial, Yeast and Fungal Samples: Microbial cultures and fungal material; genomic DNA, RNA or plasmid DNA preparation.
- 9. Stool and Soil: Stool and soil samples; DNA extraction or DNA/RNA co-extraction with inhibitor management.
- 10. Food Samples: Routine and processed solid foods, semi-solid foods and food oils.
- 11. Forensic Sample Materials: Blood stains, semen stains, hair, nails, bone and teeth.
1. Blood
These examples apply to human or other mammalian blood. Blood containing nucleated erythrocytes requires separate input and lysis guidance.
| Target / method | Representative preparation and lysis entry | Fraction and key point |
|---|---|---|
| DNA: direct whole-blood lysis | Place 20 µL Proteinase K in a tube, add 200 µL anticoagulated whole blood, then add 200 µL Buffer AL. Mix thoroughly and incubate at 70°C for 10 min.Example: D3111. | Use the whole-blood aliquot. Preliminary red blood cell (RBC) removal is unnecessary in this method. |
| DNA: buffy-coat preparation | Centrifuge anticoagulated whole blood at 2,500 × g for 10 min at RT. Collect the buffy coat between the plasma and red-cell layers. Use up to 200 µL; adjust smaller aliquots to 200 µL with PBS. Add 20 µL Proteinase K and 200 µL Buffer AL, mix and incubate at 70°C for 10 min.Example: D3111. | Retain the buffy coat. It concentrates nucleated cells, so control the cell load as well as the liquid volume. |
| Leukocyte RNA: RBC removal | Add 5 mL 1× Buffer RBC to 1 mL whole blood. Incubate on ice for 10–15 min, mixing briefly twice. Centrifuge at 400 × g for 10 min at 4°C; discard supernatant. Resuspend in 2 mL 1× Buffer RBC and repeat centrifugation. Remove liquid completely, loosen the pellet and add 0.7 mL RTL Lysis Buffer. Mix, then pass the lysate at least five times through a blunt 20-gauge needle fitted to an RNase-free syringe.Example: R4161. | Retain the leukocyte pellet. This selects leukocyte-enriched RNA. The RBC ratio, temperature and centrifugation differ from the DNA example above. |
| Whole-blood RNA: direct lysis | Place 7 mL MagZol BD3 in a 15 mL tube and add 5 mL anticoagulated blood. Immediately shake vigorously for 15 s, then vortex for 15 s. Incubate at 60°C for 10 min.Example: R4164, blood-RNA Midi B protocol. | Use the whole-blood aliquot and the dedicated blood-RNA reagent. This is the unfrozen-blood example; frozen blood has separate preparation conditions. |
2. Plasma and Serum
Prepare plasma or serum using collection and separation conditions appropriate for the intended assay. Avoid carrying over blood cells when the target is cell-free nucleic acid.
| Target / method | Representative preparation and lysis entry | Fraction and key point |
|---|---|---|
| Circulating DNA | Combine 200 µL Proteinase K, 2 mL plasma or serum, 1.6 mL Buffer ACL and 5 µL prepared Carrier RNA solution (1 µg). Mix thoroughly and incubate at 60°C for 30 min.Example: IVD3182. | Retain the liquid. A cell-pelleting method would select a different target fraction. |
| Cell-free RNA, including miRNA | After primary plasma or serum separation, centrifuge at 4,000–5,000 × g for 15 min at 4°C and transfer 1–5 mL cleared supernatant. For each 1 mL sample, add 300 µL Buffer CFL, vortex thoroughly and incubate at RT for 10–15 min. Add 100 µL Buffer CFP per 1 mL original sample, vortex vigorously for more than 20 s and place on ice for 3 min. Centrifuge at 13,000 × g for 5 min or 4,000–5,000 × g for 15 min.Example: R4316, Protocol 2. | Retain the supernatant after protein precipitation. Thorough mixing after CFP addition helps prevent nucleic acids from becoming trapped in the precipitate. For DNA removal, use the subsequent optional DNase I treatment. For combined cfDNA/RNA recovery, follow Protocol 1. |
| Viral DNA/RNA | Combine 20 µL PK/Carrier RNA with 200 µL plasma or serum. Add 400 µL Buffer VLE, vortex for 15 s and incubate at RT for 5–10 min. For smaller aliquots, bring the sample to 200 µL with 0.9% sodium chloride solution.Example: IVD4175. | Use the liquid aliquot. Thorough mixing is essential; a separate heated digestion is not specified in this example. |
| Extracellular-vesicle-associated nucleic acids | Centrifuge serum at 2,000 × g for 30 min at 4°C. Transfer 0.8 mL cleared supernatant; add 0.4 mL Exosome Precipitation Solution and incubate at 2–8°C for 30 min. Centrifuge at 10,000 × g for 10 min at RT. Remove liquid; resuspend the pellet in 100 µL PBS or sterile water. Add 20 µL Proteinase K and 300 µL Buffer GXP, mix and incubate at RT for 20 min.Example: R4319B, serum protocol. | Retain the precipitation-enriched pellet. Precipitation enriches vesicle-associated material but does not produce a purified exosome population. |
3. Animal Tissues
| Target / method | Representative preparation and lysis entry | Fraction and key point |
|---|---|---|
| Routine DNA: cut and digest | Cut <20 mg tissue into small pieces. Add 250 µL Buffer ATL and 20 µL Proteinase K. Incubate with shaking at 55°C for 30–120 min, or overnight, until digested. Add 250 µL Buffer AL, mix and incubate at 70°C for 10 min.Example: D3018. | Cutting plus digestion is sufficient for many tissues; suitable homogenization can shorten digestion. When long DNA fragments matter, control mechanical shear. |
| Routine RNA: disrupt in lysis reagent | Homogenize 10–20 mg tissue in 600 µL RTL Lysis Buffer. Alternatively, grind in liquid nitrogen and add the buffer to the powder before it thaws. Centrifuge the lysate at 14,000 × g for 3 min at RT.Example: IVD3020. | Retain the clarified lysate. Avoid homogenizing fresh tissue in PBS and leaving it unprotected before RNA lysis. |
| Insect DNA: extended digestion | Cut <10 mg insect tissue into small pieces. Add 250 µL Buffer ITL and 20 µL Proteinase K. Incubate at 55°C for 6 h or overnight, mixing periodically. If visible undigested particles remain, centrifuge at 13,000 × g for 3 min and transfer supernatant.Example: D3129. | Retain the digest or clarified supernatant. Liquid-nitrogen grinding, homogenization or bead milling can shorten digestion. For difficult material, the manual allows Proteinase K addition in two portions. |
| High-molecular-weight DNA: controlled homogenization | Slowly homogenize 10–30 mg tissue in 0.3 mL Buffer ATL Minus using a glass homogenizer and five up-and-down strokes. Transfer 0.25 mL homogenate; add 25 µL Proteinase K and 18 µL Buffer SDS. Invert to mix; incubate at 55°C for 30–120 min or until digested. If debris remains, centrifuge at 10,000 × g for 3 min and transfer 0.25 mL supernatant. Add 10 µL RNase Solution, incubate at RT for 10–30 min, then add 250 µL Buffer AL and incubate at 55°C for 10 min.Example: D6381. | Balance complete tissue disruption with preservation of long DNA fragments. Use the dedicated HMW chemistry. |
| Microbial and viral nucleic acids: fractionation | Homogenize 50–100 mg tissue in 1 mL PBS or saline. Centrifuge at 13,000 × g for 10 min. Retain the sediment for the microbial branch and transfer supernatant to a separate tube for the viral branch. Continue the selected pathogen preparation, including host-background treatment where specified.Example: IVD4179. | Keep both fractions when both branches are required. This is a fractionation step, not completed microbial lysis. It does not exclusively separate every microbe from every virus. |
4. FFPE Tissues
Deparaffinization, proteolysis and heat treatment are separate steps. The DNA and RNA schedules below belong to different protocols and should not be interchanged. Section limits also depend on tissue area and kit capacity.
| Target / method | Representative preparation and lysis entry | Fraction and key point |
|---|---|---|
| DNA: deparaffinize, digest and heat-treat | Trim excess paraffin and collect up to eight sections, 5–10 µm thick. Deparaffinize with xylene or Buffer DPS according to the selected procedure. Add 200 µL Buffer ATL and 20 µL Proteinase K to the prepared tissue. Incubate at 56°C for 60 min, then 90°C for 60 min. Add 200 µL Buffer AL and mix.Example: D3018, FFPE protocol. | Retain the deparaffinized tissue. The later high-temperature treatment helps reverse formalin-related crosslinks and modifications; it does not restore DNA already fragmented during fixation. |
| RNA: deparaffinize, digest and heat-treat | Collect up to eight sections, 5–20 µm thick. Add 600 µL Buffer DPS, vortex and incubate at 56°C for 3–5 min. Centrifuge at 14,000 × g for 2 min; discard supernatant. Add 150 µL Buffer FRL and 20 µL Proteinase K to the pellet. Incubate at 55°C for 15–30 min, then 80°C for 15 min. Add 150 µL Buffer AL and mix.Example: IVD3022. | Retain the tissue pellet after deparaffinization. Use the specified RNA heat-treatment schedule; extending it can increase RNA fragmentation. Subsequent purification includes DNase treatment. |
| Separate DNA and RNA recovery from one sample | After the IVD5116 deparaffinization procedure, add 180 µL Buffer FRL and 20 µL Proteinase K. Incubate at 56°C for 15 min, cool on ice for 3 min and centrifuge at 20,000 × g for 15 min. Transfer supernatant for RNA: incubate at 80°C for 15 min, then add 200 µL Buffer RLC. For DNA, resuspend the retained pellet in 180 µL Buffer ATL with 20 µL Proteinase K; incubate at 56°C for 1 h, then 90°C for 2 h. Add 200 µL Buffer AL.Example: IVD5116. | Retain both fractions at the separation step. The RNA supernatant and DNA pellet receive different digestion and heat treatments. |
5. Cultured Cells
| Target / method | Representative preparation and lysis entry | Fraction and key point |
|---|---|---|
| DNA: collect and resuspend | Collect <2 × 10⁶ cells at 500 × g for 5 min. Remove medium. Resuspend the pellet in 250 µL PBS with 20 µL Proteinase K; add 250 µL Buffer AL, mix and incubate at 70°C for 10 min.Example: D3018. | Retain the cell pellet. Disperse cell clumps before lysis. |
| RNA: suspension cells | Collect 3–5 × 10⁶ cells at 500 × g for 10 min. Remove medium completely, loosen the pellet and add 500–600 µL RTL Lysis Buffer. Vortex thoroughly.Example: IVD3020. | Retain the cell pellet. Proceed promptly to RNA lysis. This example does not specify a heated digestion. |
| RNA: adherent cells | Remove culture medium. For the 3–5 × 10⁶-cell preparation, add 600 µL RTL Lysis Buffer directly to the dish and pipette repeatedly to lyse and disperse the cells.Example: IVD3020. | Direct lysis avoids a separate harvesting step. Match buffer volume to cell load; a very viscous lysate may need more buffer as directed by the kit. |
6. Other Clinical Samples
6.1 Urine
| Target / method | Representative preparation and lysis entry | Fraction and key point |
|---|---|---|
| Cellular DNA | Centrifuge up to 1 mL urine at 6,000 × g for 2 min. Discard supernatant. Add 500 µL Buffer AE, vortex for 5 s and repeat centrifugation. Remove liquid; add 200 µL Buffer ATL and 20 µL Proteinase K. Incubate at 56°C for 1 h with shaking at 900 rpm. Add 200 µL Buffer AL and mix.Example: D3125. | Retain the cell pellet. If sperm cells are relevant to the DNA target, the manual allows 10 µL 1 M DTT during digestion. Cell-free DNA in discarded urine is outside this method’s target. |
| Cellular RNA | Centrifuge up to 10 mL urine at 1,600 × g for 10 min. Remove supernatant and add 500–600 µL RTL Lysis Buffer to the collected cells. Vortex thoroughly.Example: IVD3020. | Retain the cell pellet. Process promptly under the selected RNA handling conditions. |
| Cell-free DNA | Centrifuge urine at 16,000 × g for 10 min at RT. Carefully transfer the cleared supernatant to a new tube without disturbing the pellet. Use the supernatant as the starting material for cfDNA lysis and extraction.Example: IVD3182. | Retain the supernatant; discard the cell pellet. |
6.2 Amniotic Fluid
| Target / method | Representative preparation and lysis entry | Fraction and key point |
|---|---|---|
| Cellular DNA: collect and digest | Centrifuge amniotic fluid at 2,000 × g for 10 min; discard the liquid. Resuspend the cell pellet in 200 µL Buffer ATL and 20 µL Proteinase K. Incubate at 55°C for 15–20 min with shaking at 800–1,000 rpm. Add 200 µL Buffer AL, mix thoroughly and load into the corresponding automated extraction workflow.Example: IVD3101. | Retain the cell pellet. |
6.3 Saliva and Swabs
| Target / method | Representative preparation and lysis entry | Fraction and key point |
|---|---|---|
| Saliva DNA: direct liquid input | Combine 500 µL saliva with 20 µL Proteinase K and 500 µL Buffer AL. Mix thoroughly and incubate at 65°C for 30 min.Example: D3018. | Use the liquid aliquot. Saliva containing a preservation reagent needs compatibility guidance for that reagent. |
| Dry-swab DNA: digest on the swab | Place the dry swab in a 2 mL tube. Add 500 µL Buffer ATL and 20 µL Proteinase K. Incubate with shaking at 55°C for 15 min. Add 500 µL Buffer AL and mix.Example: D3018. | Keep the sample-bearing material in contact with the digest. Recover the released lysate for the kit’s purification steps. |
| Bacterial DNA: release and pellet bacteria | Add 0.5–1 mL saline or sterile water to the swab. Soak for a few minutes, vortex for 15–30 s and transfer the suspension. Collect bacteria at 10,000 × g for 5 min. Resuspend the pellet in 400 µL Buffer P1 and 20 µL kit-prepared Lysozyme; incubate at RT for 20 min. Add 400 µL Buffer DL and 20 µL Proteinase K; incubate at 70°C for 10 min. Centrifuge at 10,000 × g for 3 min.Example: D3146. | Retain the bacterial pellet before lysis and the supernatant after digestion. Difficult organisms may need the additional wall-disruption treatment in D3146. |
| Viral DNA/RNA: swab soaking liquid | Use 200 µL compatible swab soaking liquid with 20 µL PK/Carrier RNA. Add 400 µL Buffer VLE, vortex for 15 s and incubate at RT for 5–10 min.Example: IVD4175, soaking-solution application. | Use the liquid aliquot. Confirm compatibility of the swab transport medium. |
6.4 Sputum
| Target / method | Representative preparation and lysis entry | Fraction and key point |
|---|---|---|
| Pathogen RNA: liquefaction, concentration and bead disruption | Add 4–5 volumes of 0.1% DTT solution in nuclease-free water to 1–2 mL sputum. Mix and incubate at RT for 15–30 min, with repeated vortexing or shaking until clumps disappear. Centrifuge at 2,500–4,000 × g for 10 min. Remove excess supernatant, leaving approximately 500 µL liquid with the sediment, and resuspend thoroughly. Transfer the suspension to the supplied bead tube. Add 50 µL Buffer SDS and 20 µL Proteinase K. Vortex at maximum speed for 10 min, or use the specified bead-beater treatment for 60–90 s. Incubate at 55°C for 10 min, then briefly centrifuge to collect droplets. Use 250–300 µL prepared sample for purification.Example: IVD6672C. | Retain the sediment-containing suspension after concentration. DTT reduces mucus viscosity; bead disruption releases nucleic acids from difficult-to-lyse microorganisms. The 0.1% concentration refers to the added DTT solution. Subsequent DNase I treatment removes DNA for RNA recovery. |
6.5 Bronchoalveolar Lavage and Other Body Fluids
The following examples distinguish direct pathogen extraction from selective microbial DNA enrichment.
| Target / method | Representative preparation and lysis entry | Fraction and key point |
|---|---|---|
| BAL fluid: pathogen DNA/RNA | Follow the R6672B DTT liquefaction instruction for lavage fluid; its manual does not specify a DTT concentration or mixing ratio. For cell-rich material, its clarification option is 500 × g for 5 min, retaining supernatant. Add approximately 500 µL prepared sample to the bead tube with 50 µL Buffer SDS (20%). Vortex at maximum speed for 10 min, or use the specified bead grinder for 60–120 s. Centrifuge at 13,000 × g for 5 min; take 250 µL supernatant into the kit’s purification setup.Example: R6672B, lavage-fluid instructions and direct-disruption protocol. | Retain supernatant at the stated clarification steps. Low-speed clarification can exclude cell-associated targets. Proteinase K is added with the subsequent binding reagents in this kit; no separate heated predigestion is specified. |
| CSF or ascites: direct DNA extraction | Add 150 µL Buffer TL and 20 µL Proteinase K to the supplied bead tube, then add 300 µL cerebrospinal fluid (CSF) or ascites. Vortex at maximum speed for 10 min, or use the specified bead-grinder treatment for 60–90 s. Centrifuge at 13,000 × g for 3 min and transfer 250 µL supernatant for purification.Example: D3148, Protocol 1. | Use the liquid aliquot initially and retain clarified lysate after disruption. This direct-DNA route does not include selective host depletion. Host DNA may therefore be recovered along with microbial DNA. |
| CSF or ascites: microbial DNA enrichment | Centrifuge 1–1.8 mL sample at 13,000 × g for 10 min. Leave 0.7 mL liquid with the sediment and resuspend. Add 1 mL Buffer LBX1 and 5 µL Proteinase K; incubate at RT for 10 min, mixing periodically. Centrifuge at 13,000 × g for 10 min; leave approximately 50 µL liquid with the pellet and resuspend. Add 200 µL DNase Buffer and 1 µL kit-prepared DNase I (10 mg/mL); incubate at 37°C for 15 min with shaking at 600–900 rpm. Add 200 µL Buffer TL and 15 µL Proteinase K; transfer to the supplied bead tube. Vortex at maximum speed for 10 min, or use the specified bead grinder for 60–90 s. Centrifuge at 13,000 × g for 3 min; transfer 250 µL supernatant for purification.Example: D3148, Protocol 2. | Retain the microbial pellet during enrichment, then the lysate supernatant after microbial disruption. Host lysis and DNase treatment precede microbial lysis. This selects a different DNA fraction from the direct route and can lose extracellular DNA or DNA from damaged microorganisms. |
7. Plant Tissues
| Target / method | Representative preparation and lysis entry | Fraction and key point |
|---|---|---|
| Routine DNA: grind and lyse | Grind tissue in liquid nitrogen or use suitable bead disruption. For an initial trial, use 50 mg fresh/frozen tissue; D3164 allows up to 100 mg wet tissue or 20 mg dried tissue. Add 600 µL Buffer SPL and 10 µL kit RNase A solution; disperse thoroughly. Incubate at 65°C for 10 min, inverting 2–3 times. Add 200 µL Buffer PS, mix, incubate on ice for 5 min and centrifuge at >13,000 × g for 5 min. Transfer up to 600 µL supernatant.Example: D3164. | Retain the clarified lysate. Avoid tissue clumps. With liquid-nitrogen grinding, keep powder frozen until buffer addition. |
| Polyphenol-rich plant DNA: dedicated chemistry | Grind in liquid nitrogen. Begin with 50 mg fresh/frozen tissue or 15 mg dried tissue. Add 700 µL Buffer PAL, prepared with 2% PVP-40 (w/v) and 1% 2-mercaptoethanol (v/v) and preheated to 65°C. Incubate at 65°C for 15–30 min, mixing 2–3 times.Example: D3187, Protocol B. | Continue the dedicated CTAB-based extraction and clarification sequence. |
| Routine RNA: grind and lyse | Grind tissue thoroughly in liquid nitrogen and keep the powder frozen until buffer addition. Begin with ≤50 mg tissue; input may be increased to 150 mg after evaluation. Add 750 µL Buffer RLC or Buffer PRC1, prepared with 10 µL 2-mercaptoethanol or 20 µL 2 M DTT per 1 mL buffer. Vortex thoroughly and centrifuge at ≥12,000 × g for 5 min.Example: R4151. | Retain the clarified supernatant for the kit’s DNA-removal column. Use PRC1 if the sample solidifies during RLC lysis. A brief 56°C incubation for 3 min may assist PRC1 lysis, but avoid heating starch-rich samples because swelling can impair processing. |
| Broad-use RNA: low-guanidine lysis | Grind tissue in liquid nitrogen. Begin with 100 mg ordinary or polyphenol-rich tissue, or 50 mg mucilage-rich tissue; do not exceed 150 mg. Immediately add 0.9 mL Buffer RLF, prepared with 20 µL of 1 M TCEP stock per 1 mL RLF. Vortex for 10–15 s and centrifuge at 14,000 × g for 5 min at RT. Soft leaves or fruit may instead be homogenized directly in 1 mL prepared RLF; transfer 0.8–0.9 mL homogenate for clarification.Example: R4150. | Retain the supernatant for the gDNA-filter step. This low-guanidine system provides a broad-use option for unfamiliar samples and complex plant matrices. Keep frozen powder from thawing before reagent addition. |
| Difficult plant RNA: CTAB-based lysis and phase separation | Grind tissue in liquid nitrogen and transfer 50–100 mg powder to a 2 mL tube. Immediately add 700 µL Buffer PAL, vortex thoroughly and incubate at 65°C for 10 min. Where required by the sample, prepare PAL with 1% 2-mercaptoethanol (v/v); for complex polyphenol-rich material, 2% PVP-40 (w/v) may also be added. Add 700 µL Buffer BDP or chloroform, vortex for 15 s and centrifuge at 13,000 × g for 5 min at RT. Transfer approximately 500 µL upper aqueous phase.Example: R4165. | Retain the upper aqueous phase without disturbing the interphase. This CTAB-based route is intended for difficult plant matrices. The manual allows omission of PVP-40 and 2-mercaptoethanol for most samples. For applications highly sensitive to DNA contamination, use ≤50 mg tissue; subsequent purification includes DNase I treatment. |
8. Bacterial, Yeast and Fungal Samples
| Target / method | Representative preparation and lysis entry | Fraction and key point |
|---|---|---|
| Bacterial DNA: cell-wall pretreatment | Centrifuge 0.5–2 mL culture at 10,000 × g for 5 min. Remove medium. Resuspend in 400 µL Buffer P1 and 20 µL kit-prepared Lysozyme; incubate at RT for 20 min. Add 400 µL Buffer DL and 20 µL Proteinase K; mix and incubate at 70°C for 10 min. Centrifuge at 10,000 × g for 3 min.Example: D3146. | Retain bacteria before lysis and supernatant after digestion. Difficult cells may require the kit’s additional enzyme or bead treatment; lysozyme alone is not equally effective for all bacteria. |
| Bacterial RNA: enzyme pretreatment | Collect 0.5–1.8 mL exponential-phase culture (<1 × 10⁹ cells) at 12,000 × g for 1 min. Remove medium. Add 100 µL Buffer TE and 10–20 µL Lysozyme (50 mg/mL); resuspend and incubate at RT for 5–15 min. Add 500 µL Buffer RLC prepared with 2-mercaptoethanol according to the kit, vortex and incubate at RT for 5 min. Centrifuge at 12,000 × g for 1 min.Example: R4181, enzyme method. | Retain supernatant for the gDNA-filter step. This enzyme route covers Gram-negative and some Gram-positive bacteria. R4181 provides a separate bead-grinding route for difficult cells. |
| Yeast DNA: cell-wall digestion | Collect 1–1.5 mL culture at 5,000 × g for 5 min. Resuspend in 300 µL Buffer SE, 10 µL 2-mercaptoethanol and 30 µL supplied Lyticase Mixture. Incubate with shaking at 37°C for 30–60 min. Centrifuge at 5,000 × g for 5 min; discard supernatant. Resuspend in 300 µL Buffer ATL, add 300 µL Buffer DL and 10 µL Proteinase K, then incubate at 70°C for 10 min. Centrifuge at 10,000 × g for 3 min and transfer 500 µL supernatant.Example: D3147. | Retain the treated-cell pellet before lysis and clarified lysate afterward. Use the supplied enzyme mixture or match the activity of a replacement. The kit includes optional additional bead disruption. |
| Fungal RNA: mechanical disruption in reagent | Transfer <10 mg fungal material to the specified fungal/bacterial bead tube. Add 1 mL MagZol Reagent and vortex at high speed for 5–10 min. For phase separation, add 100 µL Buffer BCP, shake vigorously by hand for 15 s, stand at RT for 3 min and centrifuge at 12,000 × g for 15 min at 4°C.Example: R6623. | Retain the upper aqueous phase after separation. Disruption occurs in the RNA reagent; use the specified bead tube. |
| Plasmid DNA: controlled alkaline lysis | Harvest 25–50 mL culture at 3,000–5,000 × g for 10 min at RT. Completely resuspend the pellet in 2.5 mL Buffer E1/RNase A. Add 2.5 mL Buffer E2, invert gently 4–6 times and keep alkaline lysis to no more than 5 min. Add 2.5 mL Buffer E3 and immediately invert to mix. Centrifuge at 3,000–5,000 × g for 5 min; transfer supernatant through the kit’s lysate-clearing device.Example: P1231. | Retain the cleared liquid after neutralization. Avoid vortexing after E2 addition. |
9. Stool and Soil
| Target / method | Representative preparation and lysis entry | Fraction and key point |
|---|---|---|
| Stool DNA: bead disruption and inhibitor removal | Transfer 50–100 mg stool to the supplied bead tube with 800 µL Buffer SOL. Vortex at maximum speed for 10 min, or use the specified FastPrep-24 treatment, 6.5 m/s, twice for 45 s. Briefly spin; add 60 µL Buffer SDS and incubate at 65°C for 10 min. Centrifuge at 13,000 × g for 1 min. Transfer 600 µL supernatant; mix with 150 µL Buffer PS and 150 µL Absorber Solution, stand for 3 min and centrifuge at ≥13,000 × g for 5 min.Example: D3142, standard protocol. | Retain the final cleared supernatant. The absorber removes inhibitors but can also reduce nucleic acid recovery; the kit allows omission for certain low-nucleic-acid or lightly colored lysates. |
| Soil DNA: the same standard lysis route | Use 250–500 mg soil with 800 µL Buffer SOL in the supplied bead tube. Follow the bead disruption, 60 µL SDS / 65°C for 10 min treatment and PS/Absorber clarification described in the preceding row.Example: D3142, standard protocol. | Retain the clarified supernatant. Leave enough liquid and tube space for bead movement. The D3142 fast protocol uses a different order of SDS addition; do not mix the two sequences. |
| Environmental DNA/RNA co-extraction | Use approximately 0.5 g soil or 0.1–0.2 g stool in the specified bead tube. Add 600 µL Buffer STL and 100 µL Buffer PCI. Vortex at maximum speed for 10 min, or use the specified bead-grinder program for 30–90 s. Centrifuge at 12,000 × g for 5 min at RT. Transfer approximately 550 µL supernatant, add 200 µL Buffer SL, vortex and centrifuge at 12,000 × g for 5 min.Example: R5117B. | Retain the final supernatant. |
10. Food Samples
| Target / method | Representative preparation and lysis entry | Fraction and key point |
|---|---|---|
| Routine solid-food DNA | Transfer 200 mg representative, ground or homogenized food to a 2 mL tube. Add 800 µL Buffer ATL and 10 µL supplied Proteinase K. Incubate at 55°C for 30 min with shaking at 1,500–2,000 rpm. Add 5 µL RNase Solution; incubate at RT for 10 min. Add 400 µL Buffer PPC, vortex for 10–15 s and centrifuge at 13,000 × g for 10 min.Example: D6350, simplified food protocol. | Retain 500–800 µL supernatant. Disperse compacted material completely; avoid floating solids and the pellet during transfer. |
| Processed solid-food DNA | Grind to a fine powder. Use 100–200 mg, or ≤100 mg starch-rich material. Add 900 µL Buffer MTL and 20 µL Proteinase K (20 mg/mL); vortex for 30–60 s. Incubate at 65°C for 1–3 h, inverting occasionally. Add 300 µL Buffer PS, vortex for 30 s and place on ice for 5 min. Centrifuge at 13,000 × g for 5 min.Example: D3169, solid-food protocol. | Retain 200–600 µL supernatant for the kit’s next step. High-starch input can leave insufficient recoverable liquid. |
| Semi-solid food: sediment-based preparation | Centrifuge at 13,000 × g for 5 min. Keep a pellet of ≤0.3 mL; wash with PBS or saline if required by the matrix. Add 900 µL Buffer MTL and 20 µL Proteinase K (20 mg/mL). Incubate at 65°C for 1–3 h. Add 300 µL Buffer PS, vortex for 30 s, cool on ice for 5 min and centrifuge at 13,000 × g for 5 min.Example: D3169, semi-solid-food protocol. | Retain sediment initially, then 200–600 µL clarified lysate. This route selects target-bearing solids. Samples yielding no pellet require the alternative preparation in D3169. |
| Food oil: recover the aqueous fraction | Divide oil among four 50 mL tubes, 40 mL oil per tube. Add 2 mL Buffer ATL per tube and shake vigorously. Add 1 mL Buffer PPB per tube, mix and incubate at RT for 10 min, mixing twice. Add 3 mL Buffer PPC per tube, shake for 1 min and centrifuge at 4,000 × g for more than 20 min. Remove the upper oil phase and pool the lower aqueous phases.Example: D6350, oil protocol. | Retain the lower aqueous liquid. Avoid the oil and white interphase. |
11. Forensic Sample Materials
The sample-bearing substrate, degree of degradation and extraction target determine the preparation. Blood stains, semen stains, hair, nails, bone and teeth do not all need the same digestion chemistry.
| Target / method | Representative preparation and lysis entry | Fraction and key point |
|---|---|---|
| Blood-stain DNA: small punches | Place one to three 3 mm sample-bearing pieces in a 2 mL tube. Add 200 µL Buffer ATL and 20 µL Proteinase K. Incubate at 55°C for 60 min with shaking at 900–1,200 rpm. Add 200 µL Buffer AL; incubate at 70°C for 10 min with the same shaking. Centrifuge at 13,000 × g for 1 min.Example: D3125, blood-stain protocol. | Retain supernatant and leave the solid substrate behind. Input refers to the stained pieces, not an unrestricted amount of the carrier material. |
| Semen-stain DNA: reducing-agent-assisted digestion | Place the sample-bearing material in a 2 mL tube. Add 400 µL Buffer ATL, 4 µL 1 M DTT and 40 µL Proteinase K. Incubate at 55°C for 3–24 h with shaking at 1,000–1,500 rpm. Centrifuge at 13,000 × g for 3 min and transfer 250–300 µL supernatant.Example: D6359D, single-tube forensic protocol. | Retain the digest supernatant. DTT assists lysis of disulfide-rich sperm structures. This is a total-DNA preparation, not a differential extraction that separates sperm and non-sperm DNA. |
| Hair and nail DNA: reducing-agent-assisted digestion | Transfer hair or nail material to a 2 mL tube. Add 400 µL Buffer ATL, 4 µL 1 M DTT and 40 µL Proteinase K. Incubate at 55°C for 3–24 h with shaking at 1,000–1,500 rpm. Centrifuge at 13,000 × g for 3 min and transfer 250–300 µL supernatant.Example: D6359D, single-tube forensic protocol. | Retain supernatant. DTT supports digestion of keratin-rich material. |
| Bone and tooth DNA: powder digestion | Prepare fine powder using the specified grinding method. Transfer 100–150 mg powder; add 400 µL Buffer BGL, 4 µL 1 M DTT and 40 µL Proteinase K. Incubate at 55°C for 3–24 h with shaking at 1,000–1,500 rpm. Centrifuge at 13,000 × g for 3 min and transfer 250–300 µL supernatant.Example: D6359D, single-tube bone/teeth protocol. | Retain the digest supernatant. Fine powder improves reagent contact. |
| Bone or tooth RNA: frozen grinding | Grind under liquid nitrogen. Transfer 100–200 mg powder and immediately add 1 mL MagZol Reagent; disperse thoroughly. Add 100 µL Buffer BCP, shake by hand for 15 s and stand at RT for 3 min. Centrifuge at 12,000 × g for 15 min at 4°C.Example: R6623. | Retain the upper aqueous phase. Add the RNA lysis reagent immediately after frozen grinding. |
Adapting an unlisted sample: Match the matrix, target location and lysis requirement, then verify recovery and performance in the intended assay. Reagent names and numerical ratios are protocol-specific. Example codes identify the source method. The IVD3101 amniotic-fluid preparation is a supplied supplement, not a procedure listed in its manual.