Description
The VitroSure™ DNA Isolation Kit is designed for efficient purification of high-quality genomic DNA from frozen or fresh cells and tissue samples. Utilizing the selective binding properties of the VitroSure™ Resin Column, this kit provides a rapid, reliable, and user-friendly workflow. Up to 100 µg of highly purified genomic DNA can be isolated per preparation, suitable for a wide range of downstream molecular biology applications.
Technical Specifications
| Parameter | Specification | |
| Required Equipment | Microcentrifuge, heat block or water bath (56 °C) | |
| DNA Type | Genomic DNA | |
| Size Range | 15–30 kb | |
| Yield | Up to 100 µg total DNA (eluted in ≥50 µL) | |
| Purity | Typical A260/A280 ≥ 1.8 | |
| Storage of Eluted DNA | ≤ −20 °C | |
| Sample Types | Frozen or fresh mammalian tissues, cell pellets | |
| Input Capacity | 100 cells to 1 × 10⁷ cells; ≤30 mg tissue | |
| Downstream Applications | PCR, NGS, genotyping, restriction digestion, SNP analysis | |
Kit Contents
| Components | Volume |
| Proteinase K Powder | 5 mg × 2 |
| Proteinase K Buffer | 1 mL |
| Buffer VTL | 5 ml |
| Buffer VL | 5 mL |
| Buffer VDW1 | 15 mL |
| Buffer VDW2 | 15 mL |
| Buffer VTE | 5 mL |
| VitroSure™ DNA Elute Columns | 20 |
| Collection Tubes (2 mL) | 40 |
Storage
- Store Proteinase K powder and reconstituted Proteinase K at−20 °C.
- All remaining components may be stored at room temperature.
Procedures
Before Starting
Proteinase K Solution Preparation
- Add 260 µL Proteinase K Buffer to 5 mg Proteinase K powder.
- Vortex thoroughly until fully dissolved.
- Store the prepared solution at −20 °C.
Protocol 1: Genomic DNA Purification from Mammalian Tissue and Mouse/Rat Tail
1. Sample Preparation and Digestion
Recommended Sample Amounts
- ≤30 mg minced mammalian tissue
- ≤10 mg spleen tissue
- Mouse tail: ≤1 cm
- Rat tail: ≤0.5 cm
Tissue Disruption Recommendations
- Finely mince tissue prior to lysis.
- Grind in liquid nitrogen before adding Buffer VTL and Proteinase K.
- Homogenize tissue for improved lysis efficiency.
- For OCT-embedded tissue, section at 10–15 µm using a cryostat and remove OCT with PBS.
- For adult rodents, use 0.4–0.6 cm tail segments for optimal results.
2. Lysis
- Add 180 µL Buffer VTL and 20 µL Proteinase K. Ensure the tissue is fully submerged.
- Vortex thoroughly.
- Incubate at 56 °C for 1–4 hours, or until completely lysed. Vortex occasionally. Overnight digestion is recommended for tail samples or dense tissues.
- Centrifuge at maximum speed for 3 minutes. Transfer the supernatant to a new tube.
3. RNA Removal (Option)
If RNA-free genomic DNA is required, add 4 µl RNase A (100 mg/ml) (SKU# VB-5011, sold separately), vortex to mix, and incubate at room temperature for 2 min, then continue with step 4.
4. DNA Binding
- Add 200 µL Buffer VL, vortex for 15 seconds.
- Add 200 µL ethanol (96–100%), vortex thoroughly.
- Transfer the entire lysate to a DNA Elute Column and centrifuge at 8000 × g (≈10,000 rpm) for 1 minute. Discard flow-through.
5. Column Washing
- Add 500 µL Buffer VW1, centrifuge 1 minute; discard flow-through.
- Add 500 µL Buffer VW2, centrifuge 1 minute; discard flow-through and collection tube.
6. Membrane Drying
- Place column in a clean 2 mL tube and centrifuge at maximum speed for 3 minutes with the lid open.
7. DNA Elution
- Transfer column to a clean 1.5 mL tube.
- Apply 20–100 µL Buffer VE to the membrane center.
- Incubate for 1 minute at room temperature, then centrifuge at ≥20000 × g (≈14000 rpm) for 1 minute.
Note: Lower elution volumes increase DNA concentration but reduce total yield.
Protocol 2: Genomic DNA Purification from Blood, PBMCs, or Cultured Cells
1. Sample Preparation
- Blood with non-nucleated erythrocytes:
Add 20 µL Proteinase K to 50-100 µL blood and adjust to 220 µL with PBS. - Blood with nucleated erythrocytes:
Add 20 µL Proteinase K into 10~50 µL blood; adjust to 220 µL with PBS. - Cultured cells or PBMCs:
Pellet ≤5 × 10⁶ cells, resuspend in 200 µL PBS, and add 20 µL Proteinase K. - Frozen cell pellets:
Thaw partially and add PBS until the pellet disperses easily. Resuspend in 200 µL PBS, add 20 µL Proteinase K.
Note: For highly polyploid cells (e.g., HeLa), use fewer cells than the maximum recommended.
2. RNA Removal (Optional)
If RNA-free genomic DNA is required, add 4 µl RNase A (100 mg/ml), vortex to mix, and incubate at room temperature for 2 min, then continue with step 3.
3. Lysis
- Add 200 µL Buffer VL, vortex thoroughly, and incubate at 56 °C for 10 minutes.
4. DNA Binding
- Add 200 µL ethanol (96–100%), vortex thoroughly.
- Transfer lysate to DNA Elute Column and centrifuge at 8000 × g for 1 minute.
5. Washing and Elution
- Wash with 500 µL Buffer VDW1, then 500 µL Buffer VDW2, centrifuging 1 minute each.
- Dry membrane by centrifugation at maximum speed for 3 minutes.
- Elute DNA with 20–100 µL Buffer VTE as described in Protocol 1.
User Manual and Material Safety Data Sheet (MSDS) (PDF )
VB-5004 MSDS
Note
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
Precautions
Handle with care. Avoid contact with eyes, skin and clothing. Do not ingest. Wear gloves.
