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Description : The EZgeneTM Insect gDNA Kit is designed for efficient recovery of genomic DNA up to 60 kb in size from insects, arthropods, 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. All components of the EZgeneTM Insect gDNA Kit, except the Proteinase K and RNase A should be stored at 22oC-25oC. Once reconstituted in water, Proteinase K should be stored -20oC. Under at these conditions, DNA has successfully been purified and used for PCR after 12 months of storage. Store RNase A at 4 oC. All EZgeneTM Insect gDNA Kit components are guaranteed for at least 12 months from the date of purchase when stored at 22oC-25oC Each ezBindTM DNA column can bind approximately 100 μg DNA. Using greater than 30 mg tissue is not recommended. Product GD2413-00 GD2413-01 GD2413-02 Preps 4 50 250 ezBind DNA Columns 4 50 250 2 mL Collection tubes 8 100 500 Buffer ITL 2 mL 20 mL 100 mL Buffer BL 2 mL 20 mL 100 mL Buffer KB 2.8 mL 28 mL 135 mL Proteinase K 2 mg 30 mg 5 x 30 mg RNase A （20mg/mL） 25 μL 270 μL 1.35 mL DNA Wash Buffer 2 mL 15 mL 3 x 24 mL Elution Buffer 1 mL 15 mL 70 mL User Manual 1 1 1 Please read the entire booklet to become familiar with the EZgeneTM Insect gDNA Kit protocol. Dilute DNA Wash Buffer Concentrate with absolute ethanol as follows and store at room temperature. GD2413-00 Add 8 mL absolute (96%-100%) ethanol. GD2413-01 Add 60 mL absolute (96%-100%) ethanol to each bottle. GD2413-02 Add 96 mL absolute (96%-100%) ethanol to each bottle. Prepare proteinase K stock solution as following: GD2413-00 Add 110 μL Elution Buffer to the vial GD2413-01 Add 1.3 mL Elution Buffer to the vial GD2413-02 Add 1.3 mL Elution Buffer to each vial 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} Problem Possible Cause Suggestions Clogged Column Incomplete lysis Increase incubation time with Buffer ITL / Proteinase K. An overnight incubation may be necessary. Sample too large Do not use greater than recommended amount of starting material. For larger samples, divide into multiple tubes. Incomplete homogenization Pulverize material as indicated in liquid nitrogen to obtain a fine powder. Low DNA yield Clogged column. See above. Poor elution Repeat elution or increase elution volume. Incubate the column at 70 ℃ for 5 min before spin. Poor binding to column Follow protocol closely when adjusting binding conditions. Improper washing DNA Wash Buffer Concentrate must be diluted with ethanol before use. Low 260A/A280 ratio Extended centrifugation during elution step Resin from the column may be present in eluate. Avoid centrifugation at speeds higher than specified. The material can be removed from the eluate by centrifugation-it will not interfere with PCR or restriction digests. Poor cell lysis Increase incubation time with Buffer ITL. An overnight incubation may be necessary. Trace protein contaminants remain Following step 8, wash column with a mixture of [300 μL Buffer BL + 300 μL ethanol] before proceeding to step 9. No DNA eluted Poor cell lysis. Increase incubation time with Buffer ITL. An overnight incubation may be necessary. Incomplete homogenization Pulverize starting material as indicated in liquid nitrogen to obtain a fine powder. Absolute ethanol not added before adding sample to column Before applying DNA sample to column, add Buffer BL and absolute ethanol. No ethanol added to DNA Wash Buffer Concentrate Dilute Wash Buffer with the indicated volume of absolute ethanol before first use. Operating Protocol Introduction
Storage and Stability
Binding Capacity
Kit content
Before Starting
Determination of DNA Quality and Quantity
Trouble Shooting Guide
- Model: GD2413-01
- Manufactured by: Biomiga
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