Small amount of whole blood DNA extraction method and storage items

**RNAi Lentivirus-Mediated Service** **Screening Stable Cell Line Method** **DNA Extraction Kits** **RNA Extraction Kits** **PCR-Related Products** **DNA Marker Products** **TA Cloning Products** **Protein Research Products** **Storage Instructions:** 1. The binding solution CB or inhibitor removal solution IR may precipitate at low temperatures. To dissolve, place the vial 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 receiving, briefly centrifuge the vial and dissolve it in 1 ml of sterilized water. Due to potential loss of activity from repeated freeze-thaw cycles, store the dissolved solution in small aliquots (e.g., 20 μl) at -20°C immediately after preparation. 3. Avoid prolonged exposure to air to prevent evaporation, oxidation, or pH changes. Always close the lid tightly after each use. **Operation Steps (Please read the precautions before starting):** *Tip:* Before first use, add the specified amount of absolute ethanol to the washing buffer WB, mix well, and ensure ethanol is added to the box before use to avoid multiple additions. 1. Take 200 µl of fresh, frozen, or anticoagulated whole blood and transfer it into a 1.5 ml microcentrifuge tube. - If the sample volume is less than 200 µl, adjust to 200 µl using buffer BB. - For volumes between 200 µl and 300 µl, increase reagent amounts proportionally. - For volumes between 300 µl and 1 ml, 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 appear clear but may have a dark color. *Optional Step:* If RNA contamination is suspected, add 20 µl of RNase A (25 mg/ml) before adding CB, mix, and incubate at room temperature for 5–10 minutes. 3. After cooling, add 100 µl of isopropanol and vortex immediately. Flocs may form during this step. - Proper mixing is critical. Inadequate mixing can significantly reduce DNA yield. If the sample is sticky, vortex for 15 seconds to ensure thorough mixing. 4. Transfer the mixture (including any precipitate) into the AC column (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 washing 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 washing buffer WB, centrifuge at 12,000 rpm for 30 seconds, and discard the waste. 8. Place the AC column back into the empty collection tube and centrifuge at 13,000 rpm for 2 minutes to remove residual wash solution. This step helps prevent ethanol carryover that could inhibit downstream reactions. 9. Remove the AC column and place it in a clean microcentrifuge tube. Add 100 µl of elution buffer EB to the center of the membrane. (It is recommended to preheat the elution buffer in a 65–70°C water bath.) Let it sit for 3–5 minutes, then centrifuge at 12,000 rpm for 1 minute. Reinsert the supernatant into the column, let stand at room temperature for 2 minutes, and centrifuge again at 12,000 rpm for 1 minute. - Larger elution volumes generally improve recovery. If high DNA concentration is expected, you can reduce the elution volume, but do not go below 50 µl. Too small an elution volume may lower efficiency and yield. 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 microcentrifuge tube, or 3 ml into a 15 ml tube (available from our company). 2. Invert the anticoagulated whole blood (bring to room temperature if needed), then 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. Periodically invert the tube 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 without disturbing the white blood cell pellet. Leave about 10 µl of supernatant. - After centrifugation, white blood cell pellets should be visible at the bottom. If many red cells remain, repeat steps 3 and 4 after resuspending the pellet. 5. Resuspend the white blood cell pellet by adding 200 µl of buffer BB and vortexing. - Note: Heparinized samples may be harder to resuspend, so non-heparin anticoagulants are recommended. 6. Proceed with DNA extraction according to the standard protocol. **Product Variants:** 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 Volume Solidified Whole Blood DNA Extraction Kit – Column Type

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