E.Z.N.A.?ProtocolforTissue

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.

This method allows genomic DNA isolation from up to 30 mg tissue. Yields vary depending on source.

主要試劑

1. Warm up Elution Buffer ( 0.5 ml per sample) to 70°C.

2. Absolute ethanol - approximately 0.3 ml per sample.

3. RNase A (Optional) - stock solution at 25 mg/ml.

主要設備

1. Tabletop microcentrifuge and sterile 1.5 ml tubes.

2. Have a shaking waterbath set to 55°C.

實驗步驟

OPTIONAL: Although  no mechanical homogenization of tissue is necessary, pulverizing the  samples in liquid nitrogen will improve lysis and reduce incubation  time. Once the liquid nitrogen has evaporated, transfer the powdered  tissue to a clean 1.5 ml tube. Add 200 ul Buffer TL and proceed to step 2  below.

1. Mince up to 30 mg of tissue and place into a 1.5 ml microfuge  tube. Add 200 ul Buffer TL. Cut the tissue into small pieces to speed up  lysis. For samples larger than 30 mg, simply scale up the volume of  Buffer TL used; for a 60 mg sample use 400 ul buffer.

2. 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.

3. 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.

4. 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.

5. 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.

6. 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.

7. 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.

8. (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.

9. 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.

10. 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.

11. 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.

12. 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.

13. 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.

14. 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.