Description
VitroSure™ DNA FFPE Tissue Isolation Kit is optimized for isolation DNA from FFPE tissue samples and uses well-established VitroSure DNA Elute Columns for purification of genomic and mitochondrial DNA from small sample volume size. This kit combines the selective binding properties of a silica-based membrane with flexible elution volumes of 20-100 µl.
Kit Contents
| Buffer VTL | 5 ml |
| RNase A (100 mg/ml) | 50 µl |
| Buffer VL | 5 ml |
| Buffer VW1 | 15 ml |
| Buffer VW2 | 15 ml |
| Buffer VTE | 2 ml |
| Proteinase K Powder | 5mg×2 |
| Proteinase K Buffer | 1ml |
| VitroSure DNA Elute Columns | 20 |
| Collection Tubes (2ml) | 40 |
Technical Specifications
| Equipment needed | Microcentrifuge, heat block/bath (37°C, 56°C and 90°C) |
| DNA Type Isolated | Total DNA |
| Size Range | > 50 bp |
| Yield | Up to 25 µg total DNA can be eluted into ≥ 50 µl |
| Purity | Typical A260/A280 ≥ 1.8 |
| Eluted DNA Storage | at ≤ -20°C |
| Sample Source | Tissue from paraffin block or tissue sections |
| Processing Capacity | FFPE Tissue : ≤25 mg or 2-8 sections at thickness of 7-10 µm with a surface area of 15-20 mm2 |
| Applicable For | PCR and Next generation sequencing (NGS), genotyping, Restriction enzyme digestion, SNP, etc. |
Storage
Store the Proteinase K Powder and RNase A (100 mg/ml) at 2-8°C. After reconstitution of Proteinase K, store the solution at -20°C. The rest can be stored at room temperature.
Procedures
1. Sample preparation
1) Sample preparation from FFPE block:
- Use a scalpel to trim excess paraffin off the sample block.
- Cut up to 8 sections, each 5–10 µm thick. Discard the first 2–3 sections if the sample surface has been exposed to air.
- Place the sections immediately in a 1.5 or 2 ml microcentrifuge tube and add 1 ml of xylene to the sample. Close the lid and vortex vigorously for 10 seconds.
- Centrifuge at maximum speed for 2 minutes at room temperature.
- Carefully remove the supernatant without disturbing the pellets.
- Add 1 ml of ethanol (96–100%) to the pellet and mix by vortexing to extract residual xylene from the sample.
- Centrifuge at maximum speed for 2 minutes at room temperature.
- Carefully remove the supernatant without disturbing the pellet. Remove any remaining ethanol with a fine pipette tip.
- Open the tube and incubate at room temperature or up to 37°C for 10 minutes or until all residual ethanol has evaporated.
2) Sample preparation from FFPE sections on slides:
- Submerge the slides in xylene I for 3 minutes, followed by xylene II for an additional 3 minutes.
- Remove xylene by rinsing with 100% ethanol (1 minutes each, repeated twice).
- Air dry the slides for 3-5 minutes.
- Gently detach the tissue sections from the slides using a small blade, then transfer the tissue pellets into a 1.5 ml microcentrifuge tube.
2. Proteinase K solution preparation: Combine 260 µl of Proteinase K Storage Buffer with 5 mg of Proteinase K Powder. Vortex the mixture to ensure complete dissolution. Store the solution at -20°C.
3. Resuspend the pellets in 180 µl Buffer VTL. Add 20 µl of proteinase K solution and mix by vortexing.
4. Incubate at 56°C for 1 hour or until the sample is completely lysed.
5. Incubate at 90°C for 1 hour without agitation.
6. Cool to room temperature and briefly centrifuge the tube. For RNA-free genomic DNA, add 2 µl of RNase A (100 mg/ml) and incubate for 2 minutes at room temperature.
7. Add 400 µl of Buffer VL to the sample and mix thoroughly by vortexing. Briefly centrifuge the 1.5 ml tube.
8. Carefully transfer the entire lysate to a DNA Elute column and centrifuge at 6000×g (or 8000 rpm) for 1 minute. Discard the flow-through.
9. Add 500 µl of Buffer VW1 and centrifuge at 6000×g (or 8000 rpm) for 1 minute. Discard the flow-through.
10. Add 500 µl of Buffer VW2 and centrifuge at 6000×g (or 8000 rpm) for 1 minute. Discard the flow-through.
11. Place the DNA Elute column in a clean 2 ml collection tube. Centrifuge at maximum speed for 3 minutes with the lid open to completely dry the membrane.
12. Place the DNA Elute column in a clean 1.5 ml microcentrifuge tube and apply 20–100 µl of Buffer VTE to the center of the membrane. Ensure that Buffer VTE is at room temperature.
13. Incubate at room temperature for 5 minutes and centrifuge at maximum speed (20,000×g or 14,000 rpm) for 1 minute to elute the DNA.
User Manual and Material Safety Data Sheet (MSDS) (PDF )
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.
