实验原理

 

E.Z.N.A.TM FFPE DNA Kit uses the reversible binding properties of HiBind® matrix, a new silica-based material, combined with the speed of mini-column spin technology. A specifically formulated buffer system allows genomic DNA up to 60 kb to bind to the matrix. Samples are first lysed under denaturing conditions and then applied to the HiBind® spin columns to which DNA binds, while cellular debris, hemoglobin, and other proteins are effectively washed away. High quality DNA is finally eluted in sterile deionized water or low salt buffer.

实验步骤

 

1. Using a sclpel, trim excess paraffin off the sample block. Cut sections 10-20um thick. If the sample surface has been exposed to air, discard the first 2-3 sections.

2. Immediately transfer 3-8 sections in a 1.5 ml tube and add 200 ul Buffer TL. Vortex to mix. Incubate at 90°C for 30 minutes.

3. Sit at room temperature for 5 minutes to allow the sample to cool to room temperature before adding Protease.

4. Add 20 ul OB Protease and mix by vortexing. Then Incubate at 55°C for 1-3 hours or overnight.

5. Briefly centrifuge the tube to collect any drops from the inside of the lid. If RNAFree genomic DNA if required, add 2 ul Rnase(100mg/ml) and incubate for 5 min at room temperature.

6. Add 220 ul Buffer BL and vortex to mix.

7. Add 250 ul absolute ethanol and mix thoroughly by vortexing.

8. Assemble an HiBind® DNA MicroElute column in a 2 ml collection tube (provided). Transfer the entire sample from step 10 into the column including any precipitate that may have formed. Centrifuge at 10,000 x g for 1 min to bind DNA. Discard the collection tube and flow-through liquid.

9. Place the column into a second 2 ml collection tube and wash by pipetting 500 ul of Buffer HB. Centrifuge at 10,000 x g for 1 min. Again, dispose of collection tube and flow-through liquid.

10. Place the column into a new 2 ml collection tube and wash by pipetting 500 ul of DNA Wash Buffer diluted with ethanol. Centrifuge at 10,000 x g for 1 min. Discard flow-through.

Note: DNA Wash Buffer Concentrate must be diluted with absolute ethanol before use. See label for directions.

11. Using the same 2 ml collection tube , wash the column with a second 500 ul of DNA Wash Buffer. Centrifuge at maximum speed (10,000 x g) for 3 min to dry the column. This step is crucial for ensuring optimal elution in the following step.

12. Place the column into a sterile 1.5 ml microcentrifuge tube and add 20-100 ul of preheated (70°C) Elution Buffer. Allow tubes to sit for 3 min at room temperature.

13. To elute DNA from the column, centrifuge at 10,000 x g for 1 min. Repeat the elution with a second 20-100 ul of Elution Buffer.