Small amount of whole blood DNA extraction method and storage items

**RNAi Lentivirus-Mediated Service** **Stable Cell Line Screening Method** **DNA Extraction Kits** **RNA Extraction Kits** **PCR-Related Products** **DNA Marker Products** **TA Cloning Products** **Protein Research Products** **Storage Instructions:** 1. Binding solution CB or inhibitor removal solution IR may form precipitates at low temperatures. To dissolve, place the bottle in a 37°C water bath for a few minutes until clear and transparent. Then allow it to cool to room temperature before use. 2. Proteinase K is provided as a lyophilized powder to maintain activity and ease of transport. Upon receipt, briefly centrifuge the vial, then dissolve it in 1 ml of sterile water. Due to the risk of enzyme activity loss from repeated freeze-thaw cycles, store it at -20°C after dissolving, aliquoting into 20 μl portions for each use. 3. Avoid prolonged exposure to air to prevent evaporation, oxidation, and pH changes. Always ensure that containers are properly sealed after each use. **Operation Steps (Please read precautions before starting):** **Tip:** Before first use, add the specified amount of absolute ethanol to the wash buffer WB. Mix well and add the ethanol directly into the box to avoid multiple additions! 1. Take 200 µl of fresh, frozen, or anticoagulated blood and transfer it into a 1.5 ml microcentrifuge tube. - If the total volume is less than 200 µl, adjust with buffer BB to reach 200 µl. - If between 200–300 µl, increase reagent amounts proportionally. - If between 300–1000 µl, perform red blood cell lysis first (see appendix). 2. Add 20 µl of proteinase K (20 mg/ml), mix thoroughly. Then add 200 µl of binding solution CB, vortex, and incubate at 70°C for 10 minutes. The solution should be clear but dark in color. **Optional Step:** If RNA contamination is suspected, add 20 µl of RNase A (25 mg/ml) before adding CB. Vortex and incubate at room temperature for 5–10 minutes. 3. Cool the mixture to room temperature, then add 100 µl of isopropanol and vortex immediately. A precipitate may form. It is crucial to mix thoroughly during this step. Inadequate mixing can significantly reduce DNA yield. If the sample is sticky, vortex for 15 seconds to ensure even mixing. 4. Transfer the mixture (including any precipitate) into column AC (placed in a collection tube) and centrifuge at 13,000 rpm for 30–60 seconds. Discard the waste. 5. Add 500 µl of inhibitor removal solution IR, centrifuge at 12,000 rpm for 30 seconds, and discard the waste. 6. Add 700 µl of wash buffer WB (ensure absolute ethanol has been added), centrifuge at 12,000 rpm for 30 seconds, and discard the waste. 7. Add another 500 µl of wash buffer WB, centrifuge at 12,000 rpm for 30 seconds, and discard the waste. 8. Place column AC back into the empty collection tube and centrifuge at 13,000 rpm for 2 minutes to remove residual wash solution. This prevents ethanol from inhibiting downstream reactions. 9. Remove column AC and place it into a clean microcentrifuge tube. Add 100 µl of elution buffer EB (pre-warmed to 65–70°C if possible) to the center of the membrane. Incubate for 3–5 minutes, then centrifuge at 12,000 rpm for 1 minute. - Re-introduce the eluate back into the column, let stand at room temperature for 2 minutes, and centrifuge again for 1 minute. The larger the elution volume, the higher the DNA recovery. For high-concentration samples, you can reduce the volume, but never below 50 µl, as smaller volumes may lower recovery efficiency. 10. Store the extracted DNA at 2–8°C for short-term use or at -20°C for long-term storage. **Appendix (Example: Red Blood Cell Lysis for 300 µl – 1 ml Whole Blood):** 1. Pipette 900 µl of red blood cell lysis buffer into a 1.5 ml tube, or 3 ml into a 15 ml tube. 2. Invert the anticoagulated whole blood (allow to reach room temperature) and add 300 µl or 1 ml of blood to the respective tubes. Invert 6–8 times to mix thoroughly. 3. Incubate at room temperature for 10 minutes, gently mixing and degassing several times to aid lysis. 4. Centrifuge at 12,000 rpm for 20 seconds (for 1.5 ml tubes) or 2,000–3,000 rpm for 5 minutes (for 15 ml tubes). Discard the supernatant carefully, leaving only the white blood cell pellet and about 10 µl of supernatant. - After centrifugation, a white leukocyte pellet should be visible at the bottom. If red cells remain, repeat steps 3 and 4. 5. Resuspend the pellet by adding 200 µl of buffer BB and vortexing. - Note: Heparinized blood may be more difficult to resuspend; non-heparinized samples are recommended. 6. Proceed with DNA extraction according to the standard protocol. **Available Kits:** 1. Small Volume Whole Blood DNA Extraction Kit – Column Type 2. Small Volume Whole Blood DNA Extraction Kit – Solution Type 3. 3 ml Medium Volume Whole Blood DNA Extraction Kit (Solution Type) 4. 10 ml Large Volume Whole Blood DNA Extraction Kit (Solution Type) 5. 50 µl Small Solidified Whole Blood DNA Extraction Kit – Column Type

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