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The EZgeneTM Insect gDNA 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. Samples are homogenized and lysed in a high salt buffer and extracted with chloroform to remove polysaccharides. Following a rapid alcohol precipitation step, binding conditions are adjusted and DNA further purified using ezBindTM DNA spin columns. In this way, salts, proteins and other contaminants are removed to yield high quality genomic DNA suitable for downstream applications such as endonuclease digestion, thermal cycle amplification, and hybridization techniques.

Storage and Stability

All components of the EZgeneTM Insect gDNA Kit, except the Proteinase K and RNase A should be stored at 22 ℃-25 ℃. Once reconstituted in water, Proteinase K should be stored -20 ℃. Under at these conditions, DNA has successfully been purified and used for PCR after 24 months of storage. During shipment, or storage in cool ambient conditions, precipitates may form in some buffers. It is possible to dissolve such deposits by incubation the solution at 65℃. Store RNase A at 2-8 ℃. Expiration Date: All EZgeneTM Insect gDNA Kit components are guaranteed for at least 24 months from the date of purchase when stored at 22-25 ℃. Binding Capacity Each ezBindTM DNA column can bind approximately 100 μg DNA. Using greater than 30 mg tissue is not recommended.

Insect gDNA Isolation Protocol

Materials to be provided by user
v Microcentrifuge capable of at least 14,000 x g
v Nuclease-free 1.5 mL or 2 mL microfuge tubes
v Water bath equilibrated to 65 ℃
v Equilibrate sterile ddH2O or 10 mM Tris pH 9.0 at 65 ℃
v Absolute (96%-100%) ethanol
v Chloroform

Insect samples preserved in formalin should be rinsed in xylene and thenethanol 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 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 microtube pestle. Proceed to Step 2 below.

Arthropods (and other soft tissue invertebrates)

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 microtube pestle (Cat# SSI-1015-39 & SSI-1014-39). Addition of a pinch of white quartz sand, 50 to 70 mesh (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 ezBind column as DNA binding capacity (100 μg) may be exceeded. Meanwhile, difficult tissues may require starting with less than 30 mg tissue and doubling all volumes to ensure adequate lysis.

2. Add 350 μL Buffer ITL followed by 25 μL Proteinase K (25 mg/mL). Vortex briefly to mix and incubate at 60℃ 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 37℃ will produce adequate results.

3. To the lysate add 350 μL chloroform: isoamyl alcohol (24:1) and vortex to mix. Centrifuge at 10,000 x g for 2 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 μL of CTL1 Buffer and vortex to mix. Centrifuge as above and transfer the upper aqueous phase to tube.

4. Add one volume of Buffer BL followed by 2 μL RNase A, vortex at maxi speed for 15 s. Incubate at 70 ℃ for 10 min.

5. Add 1 volume of absolute ethanol (room temperature, 96-100%) and mix well by vortexing at maxi speed for 15 s.
TIPS: 500 μL upper aqueous solution, add 500 μL Buffer BL and 500 μL of absolute ethanol.

6. Apply 750 μL of the mixture from step 5, including any precipitation that may have formed, to the ezBind DNA column. Centrifuge at 10,000x g for 1 min at room temperature. Discard flow through liquid and re-use collection tube.

7. Place ezBind DNA column back into the same collection tube, apply the remaining of mixture into the column and centrifuge as above. Discard flow-through liquid and collection tube.

8. Place the column into another a new 2 mL collection tube (supplied) and wash by adding 500 μL KB Buffer. Centrifuge at 10,000 x g for 30 s. Discard flow-through liquid and re-use collecting tube in next step.

9. Place column into the collection tube and wash by adding 600 μL DNA Wash Buffer diluted with absolute ethanol. Centrifuge 10,000 x g for 30 s. Discard flow-through liquid and re-use collecting tube in next step.

NOTE: DNA Wash Buffer is provided as a concentrate and must be diluted with absolute ethanol as indicated on the bottle and page 4. If refrigerated, the diluted DNA wash buffer must be brought to room temperature before use.

10. Repeat step 9 with a second 600 μL DNA Wash Buffer diluted with ethanol. Discard liquid and re-insert the column to the empty collecting tube, with the lid open, and centrifuge the column at 15,000 x g for 2 min at room temperature.
NOTE: This step is critical in removing traces of ethanol that will interfere with downstream applications.

11. Place column into a clean 1.5 mL microfuge tube (not suplied). To elute DNA add 50 μL-100 μL of Elution Buffer (or 10 mM Tris buffer, pH 9.0) preheated to 60 ℃-70 ℃ directly onto the ezBind matrix. Allow soaking for 2 min at room temperature. Centrifuge at 10,000 x g for 1 min to Elute DNA.

12. Repeat elution step with a second 50 μL-100 μL Elution Buffer. Typically a total of 5-15 μg DNA with absorbance ratio (A260/A280) of 1.7-1.9 can be obtained. Yields vary depending on source and quantity of starting material used.
NOTE: To increase DNA Yield add Elution buffer and incubate the column at 60 ℃-70 ℃ for 5 min before elution.

Determination of DNA Quality and Quantity

Dilute a portion of the eluted material approximately 10-20 fold in DNA Elution Buffer or 10 mM Tris, pH 8.0. Measure absorbance at 280 nm and at 260 nm to determine the A260/ A280 ratio. Values of 1.7-1.9 generally indicate 85%-90% purity. The concentration of DNA eluted can be determined as follows: Concentration = 50 μg/mL x Absorbance260 x {Dilution Factor}