Spin Circulating DNA Kit B-HYC234-B

Components

Storage

This product can be stored at room temperature (15–25℃) for 18 months. The lyophilized

Proteinase K / Carrier RNA can be shipped and stored at room temperature. Upon receipt, it is recommended to store them at -20～-8℃. After reconstitution, Proteinase K / Carrier RNA should be kept at -20～-8℃.

Instructions

Preparations

• Absolute ethanol
• Isopropanol
• Reconstitution of Proteinase K (20 mg/mL): Add an appropriate amount of Protease Dissolve Buffer into the vial containing Proteinase K powder to a final concentration of 20 mg/mL. Invert gently to fully dissolve the lyophilized Proteinase K. The reconstituted Proteinase K should be stored at -20 ℃. Repeated freeze-thaw cycles will compromise its activity.
• Add absolute ethanol to dilute Buffer DCW2 as indicated on the bottle label, and store at room temperature.
• Add absolute ethanol to dilute Buffer DCW1 as indicated on the bottle label, and store at room temperature.
• Dilute Buffer ACB with isopropanol as indicated on the bottle label, and store at ambient temperature.
• Reconstitution of Carrier RNA (0.2 μg/μL): Add an appropriate volume of reagent to the lyophilized Carrier RNA tube according to the table below to reach a final concentration of 0.2 μg/μL. Vortex thoroughly and incubate at room temperature for 10 minutes to ensure complete dissolution. Aliquot the reconstituted Carrier RNA and store at -20 ℃.

Isolation and Storage of Plasma, Serum and Body Fluid

1. Take EDTA-anticoagulated whole blood, effusion, secretion, urine or other specimens, centrifuge at 1900 × g (3000 rpm) for 10 minutes at 4 °C to remove somatic cells. Carefully transfer the supernatant plasma or body fluid into a high-speed centrifuge tube. Approximately 4–5 mL plasma can be harvested from 10 mL whole blood.
2. Centrifuge the collected fluid at 16,000 × g for 10 minutes at 4 °C to eliminate cell debris, residual cellular nucleic acids bound to debris, as well as gDNA and RNA released from damaged blood cells.
3. Gently transfer the supernatant to a new centrifuge tube without disturbing the pellet. For same-day use, store at 2–8 °C until processing. For long-term preservation, store at −80 °C.

Thaw frozen plasma, serum or body fluid samples at room temperature prior to use. If precipitates appear after thawing, centrifuge at 16,000 × g for 5 minutes at 4 °C, then carefully transfer the supernatant to a new tube.

 Experimental Procedure

1. Add 0.5 mL Proteinase K and 5.0 mL serum, plasma or body fluid into a 50 mL centrifuge tube, then invert the tube 3–5 times to mix.
2. Add 4.0 mL Buffer ACL pre-mixed with 1 μg Carrier RNA to the sample, invert 3–5 times and vortex vigorously for 15 sec.

Prior to use, thoroughly blend Carrier RNA with Buffer ACL. The dosage of Carrier RNA per sample is 1 μg (5 μL). Carrier RNA improves DNA recovery but interferes with Qubit quantification. Its addition is optional based on experimental requirements. Sufficient vortexing at this step is required to ensure complete homogenization.

3. Incubate in a 60 °C water bath for 30 min; invert the tube occasionally during incubation.
4. Add 9.5 mL Buffer ACB, invert 6–8 times, vortex for 10 sec, then incubate on ice for 5 min.

Before use, dilute Buffer ACB with an appropriate volume of isopropanol as instructed on the bottle label or manual, and store at room temperature. Thorough vortexing is necessary to achieve full mixing at this step. 

5. Insert the Extender Tube into the HiPure CFDNA Mini Column, then fit the column assembly into the Support Tube. Place the entire connected assembly into a 50 mL centrifuge tube.

Push the Extender Tube firmly into the column to avoid liquid leakage along the wall of the Extender Tube and CFDNA column. Do not substitute with other types of 50 mL centrifuge tubes. After loading the Extender Tube, CFDNA Column and Support Tube into the 50 mL tube, a 2–3 mm protrusion will be visible. Tightly press and screw down the cap in Step 6.

6. Transfer half of the mixture from Step 4 into the Extender Tube. Press and tighten the cap firmly, then centrifuge at 3,000 × g for 5 min.
7. Carefully take out the assembly consisting of Extender Tube, CFDNA Column and Support Tube, discard the flow-through, and reinstall the whole assembly into the collection tube.
8. Transfer the remaining mixture into the Extender Tube, fasten the cap tightly, and centrifuge at 3,000 × g for 5 min.
9. Open the cap of the centrifuge tube, gently remove the column assembly, and discard the Extender Tube, Support Tube and 50 mL centrifuge tube.
10. Fit the CFDNA Mini Column onto a 2 mL collection tube, then centrifuge at 13,000 × g for 60 sec.
11. Discard the flow-through and reattach the column to the collection tube. Add 700 μL Buffer DCW1 to the column and centrifuge at 13,000 × g for 60 sec.
12. Discard the flow-through, reinstall the column on the collection tube, add 700 μL Buffer DCW2, and centrifuge at 13,000 × g for 60 sec.
13. Discard the flow-through, reinstall the column on the collection tube, add 700 μL absolute ethanol, and centrifuge at 13,000 × g for 60 sec.
14. Discard the flow-through and place the column back into the collection tube, then centrifuge at 13,000 × g for 3 min.
15. Remove the column and fit it into a new 1.5 mL collection tube, then dry the column in a 56 °C oven for 10 min.
16. Pipette 50 μL Nuclease Free Water directly onto the center of the silica membrane, incubate for 3 min, then centrifuge at 13,000 × g for 1 min.
17. Reload the collected eluate onto the membrane center, incubate for another 3 min, and centrifuge at 13,000 × g for 1 min.
18. Discard the nucleic acid binding column. Store purified DNA at −20 °C or −80 °C for long-term preservation.