HP Tissue DNA Midi Protocol

实验概要

The E.Z.N.A.^® HP Tissue DNA Midi Kit is designed for efficient recovery of genomic DNA up to 60 kb in size from up to 500mg of tissue samples. The special designed buffer system ensure the optimal lysis of tissue rich in fat, polysaccharides and fibers such as brain, adipose, muscles. This also can 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.

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 15 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 200 mg of tissue in liquid nitrogen with mortar and pestle and place the powder in a clean 15 centrifuge 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 200 mg tissue. In any event, use no more than 500 mg tissue per HiBindTM DNA Midi Column as DNA binding capacity (400 ug) may be exceeded.Meanwhile, difficult tissues may require starting with less than 200 mg tissue and doubling all volumes to ensure adequate lysis.

2. Add 3.0 ml Buffer MTL1 followed by 100 ul Proteinase K. Vortex briefly to mix and incubate at 60°C for a minimum of 2 hours or until 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 3 ml chloroform:isoamyl alcohol (24:1) and vortex to mix. Centrifuge 4,000 x g for 5 min at room temperature. Carefully transfer the upper aqueous phase to a clean 15 ml microfuge tube. Avoid the milky interface containing contaminants and inhibitors. In most case, around 2 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 the upper aqueous presented, add 1ml of Buffer MTL1 into the lysate and vortex to mix again. Centrifuge as above, then transfer the supernatant into a new 15 ml 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 50ul (assuming a sample size of 100 mg) RNase A (100 mg/ml) and incubate at room temperature for 30-60 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 sec. 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 2ml in step 3, add 2 ml Buffer BL and add 2 ml absolute ethanol.

7. Apply the mixture from step 6, including any precipitation that may have formed, to an HiBind^® DNA Midi-column assembled in a 15 ml collection tube (supplied). Centrifuge 4,000 x g for 5 min at room temperature. Discard flow-through liquid and repeat to apply the remaing mixture to the column, centrifuge as above to pass through the mixture. Discard flow-through and reuse the collection tube.

8. Place column back into the 15 ml collection tube and wash by adding 3.0 ml Buffer HB. Centrifuge 4,000 x g 2 min. Discard flow-through liquid and reuse collecting tube in next step.

9. Place column back into the 15 ml collection tube and wash by adding 3.0 ml DNA Wash Buffer diluted with ethanol. Centrifuge 4,000 x g 2 min. Discard flow-through liquid and reuse collecting 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 3.0 ml DNA Wash Buffer. Discard liquid and using the empty collection tube, centrifuge the empty column at maxi speed (no more than 8,000 x g ) for 10 min at room temperature. This step is critical in removing traces of ethanol that will interfere with downstream applications.

11. Place the HiBindTM DNA Midi column into a clean 15 ml centrifuge tube. To elute DNA add 0.5-1 ml of Elution Buffer (or 10 mM Tris buffer, pH 8.0) preheated to 60°C -70°C directly onto the HiBindTM DNA Midi column matrix. Allow to soak for 2-5 min at room temperature. Centrifuge at 6,000 x g for 5 min to collect DNA.

12. Repeat elution step with a second aliquot of Elution Buffer. Typically a total of 200 ug DNA with absorbance ratio (A260/A280) of 1.7-1.9 can be obtained from 0.5 g animal tissue. Yields vary depending on source and quantity of starting material used.