实验概要

The E.Z.N.A.^® Insect DNA Kit is designed for efficient recovery of genomic DNA up to 60 kb in size from insects, arthropods, roundworms, flatworms, and some plant tissue samples rich in polysaccharides. The method is suitable for samples frozen or preserved in alcohol or DNE solution, and good results can be obtained with formalin preserved material. 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

1. Absolute (96%-100%) ethanol

2. Chloroform:isoamyl alcohol (24:1)

主要设备

1. Microcentrifuge capable of at least 10,000 x g

2. Nuclease-free 1.5 ml or 2 ml microfuge tubes

3. Water bath equilibrated to 60°C

实验步骤

Insect 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 analysis.

Insects:

1. Pulverize no more than 50 mg of tissu e in liquid nitrogen with mortar and pestle and place the powder in a clean 1.5 ml microcentrifuge tube. If ceramic mortar and pestle are not available, homogenize the sample in the microfuge tube using a disposable microtube pestle (Omega Bio-Tek, Cat Cat# SSI-1015-39 & SSI-1014-39).Proceed to Step 2 below.

Arthropods (and other soft tissued invertebrates and plant samples):

1. Grind no more than 30 mg tissue in liquid nitrogen with mortar and pestle and place the powder in a clean 1.5 ml microcentrifuge tube. If ceramic mortar and pestle are not available, homogenize the sample in the microfuge tube using a disposable m icrotube pestle (Cat# SSI-1015-39 & SSI-1014-39). Addition of a pinch of white quartz sand, -50 to 70 m esh (Sigma Chemical Co. Cat No. S9887) will help. Proceed to Step 2 below.

Amount of starting material depends on sample and can be increased if acceptable results are obtained with the suggested 30 mg tissue. For easy to process specimens, the procedure may be scaled up and the volumes of all buffers used increased in proportion. In any event, use no more than 50 mg tissue per HiBind^® spin-column as DNA binding capacity (100 ug) may be exceeded. Meanwhile, difficult tissues may require starting with less than 30 mg tissue and doubling all volum es to ensure adequate lysis.

2. Add 350 ul Buffer CTL followed by 25 ul Proteinase K (20 mg/ml). Vortex briefly to mix and incubate at 60°C for a minimum of 30 min or until entire sample is solubilized. Actual incubation times vary and depend on elasticity of tissues. Most samples require no more than 4 hours. Alternatively an overnight incubation at 55°C will produce adequate results.

OPTIONAL: Certain tissues have high levels of RNA which will be co-purified with DNA using this kit. W hile 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 tem perature for 10-15 m inutes. Proceed with the protocol.

3. To the lysate add 350 ul chloroform:isoamyl alcohol (24:1) and vortex to mix. Centrifuge at 10,000 x g for 5 min at room temperature. Carefully transfer the upper aqueous phase to a clean 1.5 ml microfuge tube. Avoid the milky interface containing contaminants and inhibitors.

Note: This step will remove much of the polysaccharides and proteins from solution and improve spin-column performance downstream. If very few upper aqueous phase present after centrifugation, add 200 ul of Buffer CTL and vortex to mix. Centrifuge as above and transfer the upper aqueous phase to tube.

4. Add one volume of Buffer CBL and vortex at maxi speed for 15s. Incubate at 60°C for 10 m inutes.

5. Add one volume of absolute ethanol (room temperature, 96-100%) and mix well by vortexing at maxi speed for 15s. Tips: 250 ul upper aqueous solution, add 250 ul Buffer CBL and 250 ul of absolute ethanol.

6. Apply 750 ul of the mixture from step 5, including any precipitation that may have formed, to a HiBind^® DNA column assembled in a 2 ml collection tube (supplied). Centrifuge at 10,000 x g for 1 m in at room tem perature. D iscard flow-through liquid and re-use collection tube.

7. Place HiBind^® DNA column back into the same collection tube, apply the rem aining of mixture into the column and centrifuge as above. Discard flow-through liquid and collection tube.

8. Place the column into the same collection tube and wash by adding 500 ul Buffer HB. Centrifuge at 10,000 x g for 1min. Discard the flow-through and collection tube.

9. Place column into a new 2 ml collection tube (supplied) and wash by adding 700 ul DNA Wash Buffer diluted w ith absolute ethanol. Centrifuge 10,000 x g for 1 min as above. Discard flowthrough liquid and re-use collecting 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. If refrigerated, the diluted DNA wash buffer must be brought to room temperature before use.

10. Place column into a new 2 ml collection tube (supplied) and wash by another 700 ul DNA Wash Buffer diluted with ethanol. Centrifuge as above and discard liquid.

11. Place the empty column back into the same collection tube. Centrifuge the column at 10,000 x g for 2 min at room temperature. This step is critical in removing traces of ethanol that will interfere with downstream applications.

12. Place column into a clean 1.5 ml microfuge tube (not supplied). To elute DNA, add 50-100 ul of Elution Buffer preheated to 60°C directly onto the HiBind^® matrix. Allow to soak for 2 min at room temperature. Centrifuge at 10,000 x g for 1 min to Elute DNA.

13. Optional: Repeat elution step with a second 50-100 ul Elution Buffer.

Typically a total of 5-50 ug DNA with absorbance ratio (A260/A280) of 1.7-1.9 can be obtained from 30 mg tissue sample. Yields vary depending on source and quantity of starting material used.

TIP: To increase DNA Yield add Elution buffer and incubate the column at 60°C-70°C for 5 min before elution.