產品介紹

The E.Z.N.A.? FFPE DNA Kit is designed for fast and easy purification of DNA from formalin-fixed, paraffin-embedded (FFPE) tissue sections. Paraffin removal can be performed using a xylene and ethanol method or efficient heat treatment. Samples are incubated in a specialized lysis buffer along with Proteinase K to reverse crosslinking, effectively releasing short and long DNA fragments. After adjusting the binding conditions with ethanol, the lysate is applied to the MicroElute? DNA column to bind DNA. Cellular debris and proteins are effectively removed during the wash steps. High quality purified DNA is then eluted in elution buffer or water and is ready for applications such as PCR and next-generation sequencing.

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流程

DNA Extraction and Purification from FFPE – Xylene Method

1. Add 1 mL xylene to a 1.5 mL or 2 mL microcentrifuge tube.

2. Cut 3-8 paraffin sections 5-10 μm thick. Do not use the first 2-3 sections. Immediately place the section(s) into the xylene. Vortex for 20 seconds to mix thoroughly.

3. Centrifuge at maximum speed for 2 minutes. Aspirate and discard the supernatant. Do not disturb the pellet.

4. Add 1 mL 100% ethanol. Vortex to mix thoroughly.

5. Centrifuge at maximum speed for 2 minutes. Aspirate and discard the supernatant. Do not disturb the pellet.

6. With the lid open, dry the pellet at 37°C for 15 minutes. Carefully aspirate any residual ethanol with a pipettor before proceeding to the next step.

7. Add 200 μL FTL2 Buffer and pipet up and down to resuspend the pellet.

8. Add 20 μL Proteinase K Solution. Vortex to mix thoroughly.

9. Incubate at 55°C for 3 hours. Incubation can proceed overnight.

10. Incubate at 90°C for 10-30 minutes. Centrifuge the tube briefly to collect any liquid adhering to the lid.
    Optional: If RNA-free gDNA is required, add 10 μL RNase A (20 mg/mL, not provided) and let sit for 5 minutes at room temperature.

11. Add 220 μL BL Buffer. Vortex to mix thoroughly.

12. Add 250 μL 100% ethanol. Vortex to mix thoroughly.

13. Insert a MicroElute? LE DNA Column into a 2 mL Collection Tube (provided) and follow the column equilibration steps listed below:

Protocol for Column Equilibration:

1. Add 100 μL 3M NaOH to the MicroElute? LE DNA Column.

2. Centrifuge at 10,000 x g for 30 seconds.

3. Add 500 μL sterile deionized water to the MicroElute? LE DNA Column.

4. Centrifuge at 10,000 x g for 30 seconds.

5. Discard the filtrate and reuse the collection tube.

 14. Transfer the entire sample from Step 12 (including any precipitates) to the MicroElute? LE DNA Column.

 15. Centrifuge at 10,000 g for 1 minute. Discard the filtrate and the collection tube.

 16. Transfer the MicroElute? LE DNA Column to a new 2 mL Collection Tube.

 17. Add 500 μL HBC Buffer diluted with 100% isopropanol (see the bottle for instructions). Centrifuge at 10,000g for 1 minute. Discard the filtrate and the collection tube.

 18. Transfer the MicroElute? LE DNA Column to a new 2 mL Collection Tube.

 19. Add 700 μL DNA Wash Buffer diluted with 100% ethanol (see the bottle for instructions). Centrifuge at 10,000g for 1 minute. Discard the filtrate and reuse the collection tube.

 20. Repeat Step 19 for a second DNA Wash step.

 21. Centrifuge the empty MicroElute? LE DNA Column at maximum speed for 2 minutes to dry the column. This step is critical for removal of trace ethanol that may interfere with downstream applications.

 22. Place the MicroElute? LE DNA Column into a new 1.5 mL microcentrifuge tube.

 23. Add 50-75 μL Elution Buffer heated to 70°C. Let sit for 3 minutes at room temperature. Centrifuge at maximum speed for 1 minute.

 24. Repeat Step 23 for a second elution step.

 25. Store eluted DNA at -20°C.

DNA Extraction and Purification – Heat Method

1. Add 200 μL FTL2 Buffer into a 1.5 mL or 2 mL microcentrifuge tube (not provided).

2. Cut 3-4 paraffin sections 5-10 μm thick. Do not use the first 2-3 sections. Immediately place the section(s) into the FTL2 Buffer. Vortex for 20 seconds.

3. Incubate at 90°C for 15 minutes. Gently shake the tube 2-3 times. Make sure that the sections stay submerged.

4. Let sit at room temperature for 5 minutes to cool before adding Proteinase K Solution. If the sample temperature is too high, Proteinase K can be inactivated.

5. Add 20 μL Proteinase K Solution. Vortex to mix thoroughly.

6. Incubate at 55°C for 3 hours. Incubation can proceed overnight.

7. Centrifuge the tube briefly to collect any liquid adhering to the lid.
   Optional: If RNA-free gDNA is required, add 10 μL RNase A (20 mg/mL, not provided) and let sit for 5 minutes at room temperature.

8. Proceed to Step 11 of the DNA EXTRACTION AND PURIFICATION - XYLENE METHOD protocol on the reverse page.