E.Z.N.A.?ProtocolforParaffinEmbeddedTissue

實驗概要

The E.Z.N.A.^? Tissue DNA Kit provides a rapid and easy method for the isolation of genomic DNA for consistent PCR and Southern analysis. Up to 30 mg tissue or up to 1 cm sections of mouse tail can be readily processed in one time. The method can also be used for preparation of genomic DNA from mouse tail snips, blood, buffy coat, serum, and plasma. The kit allows single or multiple, simultaneous processing of samples. There is no need for phenol/chloroform extractions, and timeconsuming steps such as precipitation with isopropanol or ethanol, are eliminated. DNA purified using the E.Z.N.A.^? Tissue DNA method is ready for applications such as PCR*, Southern blotting, and restriction digestion.

實驗步驟

1. Place not more than 30 mg tissue (~2 mm³) in a clean 2 ml microfuge tube.

2. Extract the sample with 1 ml xylene to remove the paraffin. Mix thoroughly by vortexing.

3. Centrifuge the tube at 10,000 x g for 10 min at room temperature. Discard supernatant without disturbing the tissue pellet.

4. Rinse the pellet with 1 ml absolute ethanol to remove traces of xylene. Centrifuge at 10,000 x g for 5 min at room temperature. Discard the ethanol without disturbing the tissue pellet.

5. Repeat the ethanol rinse.

6. Air dry tissue pellet at 37°C for 15 min.

7. Add 200 ul Buffer TL to the tissue.

8. Add 25 ul of OB Protease and vortex to mix well. Incubate at 55°C in a shaking waterbath to effect complete lysis. If no shaking waterbath is available, vortex the sample every 20-30 minutes. Lysis time depends on amount and type of tissue, but is usually under 3 hours. One can allow lysis to proceed overnight.

The volume of OB Protease (or proteinase K) used will need to be adjusted based on amount of starting material; use 50 ul for a 60 mg tissue sample.

9. OPTIONAL: Certain tissues such as liver have high levels of RNA which will be copurified with DNA using this kit. While it will not interfere with PCR, the RNA may be removed at this point. Add 5ul (assuming a sample size of 30 mg) RNase A (25 mg/ml) and incubate at room temperature for 2-5 minutes. Proceed with the tissue protocol.

10. Centrifuge for 5 min at 10,000 x g to pellet insoluble tissue debris. Carefully aspirate the supernatant and transfer to a sterile micro-centrifuge tube leaving behind any insoluble pellet.

11. Add 220 ul Buffer BL and vortex to mix. Incubate at 70°C for 10 min. A wispy precipitate may form on addition of Buffer BL, but does not interfere with DNA recovery. Adjust the volume of Buffer BL required based on amount of starting material.

12. Add 220 ul absolute ethanol (room temperature, 96-100%) and mix thoroughly by vortexing at maxi speed for 15 seconds. Adjust the volume of ethanol if greater than 30 mg tissue is used). If precipitation can be seen at this point, break the precipitation by pipetting up and down 10 times.

13. Assemble a HiBind?DNA column in a 2 ml collection tube (provided). Transfer the entire lysate from step 6 into the column including any precipitate that may have formed. Centrifuge at 8,000 x g for 1 min to bind DNA. Discard flow-through liquid.

14. (Optional) If greater than 30 mg tissue is used, repeat transfer the remaining lysate into the column and centrifuge as above. Make sure that all of the lysate has pass through the column.

15. Place the column into a second 2 ml collection tube and wash by pipetting 500 ul of Buffer HB. Centrifuge at 8,000 x g for 1 min. Discard flow-through liquid and 2ml collection tube.

16. Place the column into a second 2 ml collection tube and wash by pipetting 700 ul of DNA Wash Buffer diluted with ethanol. Centrifuge at 8,000 x g for 1 min. Discard flow-through liquid and re-use 2ml collection tube in next step.

Note that DNA Wash Buffer is provided as a concentrate and must be diluted with absolute ethanol as indicated on the bottle or page 3. If refrigerated, the diluted DNA wash buffer must be brought to room temperature before use.

17. Place the column back into the 2ml collection tube from step 10, wash the column with a second 700 ul of DNA Wash Buffer diluted with ethanol and centrifuge as above. Discard flow-through.

18. Place the column back into the same 2 ml collection tube, centrifuge the empty column at maximum speed (>12,000 x g) for 2 min to dry the column. This step is crucial for ensuring optimal elution in the following step.

19. Place the column into a sterile 1.5 ml microfuge tube and add 50-200 ul of preheated (70°C) Elution Buffer. Allow tubes to sit for 3 min at room temperature.

20. To elute DNA from the column, centrifuge at 10,000 x g for 1 min. Repeat the elution with a second 100-200 ul of Elution Buffer.

Note: Each 100-200 ul elution typically yields 60-70% of the DNA bound to the column. Thus two elutions generally give ~90%. However, increasing elution volume reduces the concentration of the final product. To obtain DNA at higher concentrations, elution can be carried out using 50 ul to 100 ul Elution Buffer (which slightly reduces overall DNA yield). Volumes lower than 50 ul greatly reduce yields. In some instances yields may be increased by incubating the column at 70°C (rather than at room temperature) upon addition of Elution Buffer. If necessary the DNA can be concentrated. Add sodium chloride to a final concentration of 0.1 M followed by 2X volume of absolute (100%) ethanol. Mix well and incubate at -20°C for 10 min. Centrifuge at 10,000 x g for 15 min and discard supernatant. Add 700 ul of 80% ethanol and centrifuge at 10,000 x g for 2 min. Discard supernatant, air dry the pellet (2 min) and resuspend DNA in 20 ul sterile deionized water or 10 mM Tris-HCl, pH 8.0.

Yields will depend on size and age of sample. Certain samples may require prolonged lysis with Buffer TL.

Note: Tissue fixed with paraformaldehyde will yield degraded DNA or RNA. The extent of degradation depends on type of fixative used, but the size of DNA obtained is usually less than 500 bp. Degradation is not caused by the E.Z.N.A.^? Tissue DNA protocol, and for PCR detection of segments smaller than 500 bp satisfactory results can be obtained.