HPTissueDNAMaxiProtocol

实验概要

The E.Z.N.A.^®  HP Tissue DNA Maxi Kit is designed for efficient recovery of genomic  DNA up to 60 kb in size from up to 2 grams of tissue samples. The  special designed buffer systems ensure the optimal lysis of tissue rich  in fat, polysaccharides and fibers such as brain, adipose, muscle. This  Kit can also isolate DNA from molluscs, insects, arthropods, roundworms,  flatworms, and other invertebrate tissue samples rich in  mucopolysaccharides. The procedure relies on the well-established  properties of the cationic detergent, cetyltrimethyl ammonium bromide  (CTAB), in conjunction with the selective DNA binding of Omega Bio-Tek’s  HiBind^® matrix.

Samples are homogenized and lysed in a high salt buffer containing  CTAB and digested with proteinase. After addition of chloroform, the  homogenate is separate into aqueous and organic phases by  centrifugation. The upper, aqueous phase is extracted and buffer BL is  added to provide appropriate binding conditions. The sample is then  loaded into the HiBind^® DNA Maxi Spin Column, where the  genomic DNA binds to the membrane and salt and other contaminants are  efficiently washed way. High quality genomic DNA is then eluted with  Elution buffer or water. Purified DNA is suitable for most downstream  applications such as endonuclease digestion, thermal cycle  amplification, and hybridization techniques.

主要试剂

Regents to be Provided by User

1. Absolute ethanol (96-100%)

2. Sterile deionized water

3. Chloroform - prepare Chloroform:isoamyl alcohol (24:1)

主要设备

Equipments to be Provided by User

1. Laboratory centrifuge equipped with swinging-bucket rotor capable of 2000-5000 × g.

2. Sterile 50 ml microfuge tubes

3. Water bath equilibrated to 60°C

实验步骤

Samples preserved  in formalin should be rinsed in xylene and then ethanol before  processing. Note that results obtained with formalin-fixed tissues  generally depend on age and size of specimen. Purified material is  usually adequate for PCR amplification, but fresh or frozen samples  should be used for southern analyses.

1. Pulverize 500 mg of tissue in liquid nitrogen with mortar and  pestle and place the powder in a clean 50ml tube. Sample can also be  ground and homogenized by beads mill. Amount of starting material  depends on sample and can be increased if acceptable results are  obtained with the suggested 500 mg tissue. In any event, use no more  than 2 grams of tissue per HiBindTM DNA Maxi Column as DNA binding  capacity (2.5 mg) may be exceeded. Meanwhile, difficult tissues may  require starting with less than 500 mg tissue and doubling all volumes  to ensure adequate lysis.

2. Add 9.0 ml Buffer MTL1 followed by 130 ul Proteinase K. Vortex  briefly to mix and incubate at 60°C for a minimum of 2 hours or until  the entire sample is solubilized. Actual incubation time varies and  depends on elasticity of tissue. Most samples require no more than 4  hours. Alternatively an overnight incubation at 55°C will produce  adequate results.

3. To the lysate add 9 ml chloroform:isoamyl alcohol (24:1) and  vortex to mix at maxi speed for 15 seconds. Centrifuge 4,000 x g for 10  min at room temperature. Carefully transfer the upper aqueous phase to a  clean 50 ml microfuge tube. Avoid the milky interface containing  contaminants and inhibitors. In most case, around 6 ml upper phase can  be transferred.

Note: This step will remove much of the polysaccharides and proteins  from solution and improve spin-column performance downstream. If very  few upper aqueous solution presented, add 2 ml Buffer MTL1 and vortex to  mix. Centrifuge as above and transfer the upper aqueous solution into a  new tube.

4. OPTIONAL: Certain tissues such as liver have high levels of RNA  which will be co-purified with DNA using this kit. While it will not  interfere with PCR, the RNA may be removed at this point. Add 30ul  (assuming a sample size of 500 mg) RNase A (25 mg/ml) and incubate at  room temperature for 15-60 minutes. Proceed with the tissue protocol.

5. Add equal volume of Buffer BL and vortex to mix. Incubate at 70°C  for 10 minutes. A wispy precipitate may form on addition of Buffer BL,  but does not interfere with DNA recovery.

6. Add equal volume of absolute ethanol (room temperature, 96-100%)  and mix throughly by vortexing at maxi speed for 30 seconds. If  precipitation can be seen at this point, break the precipitation by  passing through a needle using a syringe.

Tip: For example if the total upper aqueous phase volume is 6ml in step 3, add 6 ml Buffer BL and add 6 ml absolute ethanol.

7. Apply the mixture from step 6, including any precipitation that may have formed, to an HiBind^®  DNA Maxi-column assembled in a 50 ml collection tube (supplied).  Centrifuge 4,000 x g for 5 min at room temperature. Discard flowthrough  liquid. Repeat to apply the remaining mixture to column and centrifuge  as above. Discard flow-through liquid.

8. Place column back into the 50 ml collection tube. Add 9 ml Buffer  HB and centrifuge 4,000 x g for 5 min as above. Discard flow-through  liquid and reuse the collecting tube in the next step.

9. Place column into 50 ml collection tube and wash by adding 12 ml  DNA Wash Buffer diluted with absolute ethanol. Centrifuge 4,000 x g for 5  min as above. Discard flow-through liquid and reuse collection tube in  next step.

Note: DNA Wash Buffer is supplied as a concentrate and must be  diluted with absolute ethanol as indicated on page 4 of this booklet.

10. Repeat step 9 with a second 10 ml DNA Wash Buffer diluted with  absolute ethanol. Discard liquid and using the empty collection tube,  centrifuge the column at 4,000 x g for 15 min at room temperature. This  step is critical in removing traces of ethanol that will interfere with  downstream applications (such as agarose gel electrophoresis of high  molecular weight DNA).

11. Place HiBind^® DNA Maxi-column into a clean 50 ml  centrifuge tube. To elute DNA add 3-4ml of Elution Buffer (or 10 mM Tris  buffer, pH 8.0) preheated to 60°C -70°C directly onto the HiBindTM DNA  Maxi columne matrix. Allow to soak for 5-10 min at room temperature.  Centrifuge at 4,000 x g for 5 min to collect DNA.

12. Repeat elution step with a second aliquot of Elution Buffer.  Typically a total of 400 ug DNA with absorbance ratio (A260/A280) of  1.7-1.9 can be obtained from 0.5 gram animal tissue. Yields vary  depending on source and quantity of starting material used.