Spin Circulating DNA Mini Kit-HYC231
Circulating DNA is extracellular free DNA generated during cell apoptosis, with fragment sizes generally less than 1 kb. Adopting silica-column purification technology, this extraction procedure eliminates toxic phenol-chloroform extraction and time-consuming ethanol precipitation. The recovered circulating DNA can be directly applied to quantitative PCR, liquid/solid phase microarray analysis, hybridization, SNP detection and other downstream analyses. This reagent enables efficient capture of trace cell-free nucleic acids from up to 1 mL plasma.
Components
|
Components |
50 T |
250 T |
|
Buffer ACL |
50 mL |
250 mL |
|
Buffer DCW1 * |
13 mL |
66 mL |
|
Buffer DCW2 * |
20 mL |
50 mL |
|
Elution Buffer |
15 mL |
30 mL |
|
Carrier RNA |
120 µg |
310 µg |
|
Proteinase K |
120 mg |
5×120 mg |
|
Protease Dissolve Buffer |
15 mL |
60 mL |
|
Viral Mini Columns |
50 |
250 |
|
2ml Collection Tube |
100 |
5×500 |
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.
- 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 ℃.
|
Components |
50 T |
250 T |
|
Carrier RNA |
120 µg |
310 µg |
|
Buffer AE |
500 µL |
1.4 mL |
|
Proteinase K |
5 µL |
20 µL |
Isolation and Storage of Plasma, Serum and Body Fluid
- Take samples such as EDTA-anticoagulated blood, effusion and secretion. Centrifuge at 4 °C, 1900 × g (3000 rpm) for 10 minutes to remove somatic cells. Carefully transfer the collected plasma or body fluid into a high-speed centrifuge tube.
Generally, about 4–5 mL of plasma can be obtained from 10 mL of whole blood.
- Centrifuge at 4 °C, 16,000 × g for 10 minutes to eliminate cell debris, excess cellular nucleic acids attached to cell debris, as well as gDNA and RNA released from damaged blood cells.
- Carefully transfer the supernatant to a new centrifuge tube without aspirating the pellet.
Samples to be used on the same day can be stored at 2–8 °C until use. For long-term storage, keep samples at −80 °C.Frozen plasma, serum or body fluid samples shall be thawed completely at room temperature before use. If precipitate appears after thawing, centrifuge at 4 °C, 16,000 × g for 5 minutes, then carefully transfer the supernatant to a new centrifuge tube.
Experimental Procedure
- Transfer 100 μL Proteinase K into a 5.0 mL centrifuge tube, then add 1.0 mL plasma, serum, urine, effusion or other cell-free body fluid samples. Invert the tube 3–5 times to mix thoroughly.
- Add 1.0 mL Buffer ACL and (optional) 5 μL Carrier RNA to the sample, then invert 15–20 times for full mixing.
Prior to use, pre-mix Carrier RNA with Buffer ACL. The recommended 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.
- Incubate in a 60 °C water bath for 30–60 min; invert the tube several times every 10–15 min during incubation.
- Add 0.7 mL isopropanol to the sample, invert 3–5 times, vortex for 10–15 sec, then incubate on ice for 3–5 min.
- Fit a Viral Mini Column onto a 2 mL collection tube, load 750 μL mixture onto the column, and centrifuge at 10,000 × g for 1 min.
- Discard flow-through and reinstall the column onto the collection tube. Load another 750 μL mixture and centrifuge at 10,000 × g for 1 min. Repeat this step until the entire mixture has passed through the column.
- Mount the column onto a new collection tube. Add 500 μL ethanol-diluted Buffer DCW1 to the column and centrifuge at 10,000 × g for 1 min.
- Discard flow-through and reattach the column to the collection tube. Add 500 μL ethanol-diluted Buffer DCW2 to the column and centrifuge at 10,000 × g for 1 min.
- Discard flow-through and reattach the column to the collection tube. Add another 500 μL ethanol-diluted Buffer DCW2 to the column and centrifuge at 10,000 × g for 1 min.
- Discard flow-through, place the column back into the collection tube, then centrifuge at 13,000 × g for 2 min.
- Transfer the column to a new 1.5 mL centrifuge tube and dry the column further at 56 °C for 10 min.
- Pipette 40–50 μL Elution Buffer directly onto the center of the silica membrane, incubate for 2 min, then centrifuge at 13,000 × g for 1 min.
- Reload the collected eluate onto the membrane center, incubate for another 2 min, and centrifuge at 13,000 × g for 1 min.
- Discard the nucleic acid binding column. Store purified DNA at 2–8 °C for short-term use, or at −20 °C for long-term preservation.