Contents Introduction Storage and Stability
Contents
1. proteinase K stock solution as following Buffer HB 3 ml 30 ml 110 ml DNA Wash Buffer Concentrate 2 ml 20 ml 3 x 20 ml D3373 00 Add 150 ul Elution Buffer to the vial Elution Butter 1ml 20 mi somi D3373 01 Add 1 5 ml Elution Buffer to the vial User Manual 1 1 SSS D3373 02 Add 1 5 ml Elution Buffer to each vial Vortex vial briefly prior to use We recommend that you aliquot and store vials of reconstituted protease at 20 C Materials to be provided by user e Microcentrifuge capable of at least 14 000 x g o Nuclease free 1 5 ml or 2 ml microfuge tubes e Water bath equilibrated to 65 C Equilibrate sterile dH O or 10 mM Tris pH 9 0 at 65 C e Absolute 96 100 ethanol o Chloroform E Z N A Mollusc Arthropod DNA Protocol Invertebrates 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 Arthropods 1a 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 Omega Bio Tek Cat No SSI 1015 39 Eppendorf Cat No 0030 120 973 VWR Cat2. the E Z N A Mollusc DNA Kit except the Proteinase K and RNase A should be stored at 22 C 25 C Once reconstituted in water Proteinase K should be stored at 20 C Store Rnase A at 20 C 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 C Store RNase A at 20 C Expiration Date All E Z N A Mollusc DNA Kit components are guaranteed for at least 24 months from the date of purchase when stored at 22 25 C Binding Capacity Each HiBind DNA column can bind approximately 100 ug DNA Using greater than 30 mg tissue is not recommended Kit Contents Before Starting Lats a See See Please read the entire booklet to become familiar with the E Z N A Product D3373 00 D3373 01 D3373 02 Mollusc DNA Kit protocol Purification times 5 Preps 50 Preps 200 ma Dilute DNA Wash Buffer Concentrate with absolute ethanol as follows and HiBind DNA Columns 5 50 store at room temperature 2 ml Collecting tubes 10 100 D3373 00 Add 8 ml absolute 96 100 ethanol Buffer ML1 2 ml 20 ml 80 ml Buffer MBL 25ml 25 ml 100 ml D3373 01 Add 80 ml 96 100 ethanol to each bottle Proteinase K 3 mg 30 mg 4 x 30 mg D3373 02 Add 80 ml 96 100 ethanol to each bottle RN A 0 ul 270 ul 1 1 ml pags PH p a Prepare
3. Contents Introduction Storage and Stability Kit Contents Materials to Be Provided by User 0 0 cee eee eee Before Starting Mollusc Arthropod DNA Protocol 0000020 cece eeeee Determination of DNA Quality and Quantity Troubleshooting Guide Introduction The E Z N A Mollusc DNA Kit is designed for efficient recovery of genomic DNA up to 60 kb in size from molluscs insects arthropods roundworms flatworms and other invertebrate tissue samples rich in mucopolysaccharides The method is suitable for invertebrates 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 Samples are homogenized and lysed in a high salt buffer containing CTAB and extracted with chloroform to remove mucopolysaccharides Following a rapid alcohol precipitation step binding conditions are adjusted and DNA further purified using HiBind DNA Mini 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
4. No KT 749520 0000 Proceed to step 2 below Molluscs and other soft tissued invertebrates 1b 2 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 amp SSI 1014 39 Addition of a pinch of white quartz sand 50 to 70 mesh Sigma Chemical Co Cat No 9887 will help Proceed with 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 DNA column as binding capacity 100 ug may be exceeded Meanwhile difficult tissues may require starting with less than 30 mg tissue and doubling all volumes to ensure adequate lysis Add 350 ul Buffer ML1 followed by 25 ul Proteinase K Vortex to mix and incubate at 60 C for a minimum of 30 min 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 37 C will produce adequate results To the lysate add 350 ul chloroform isoamyl alcohol 24 1 and vortex to mix Centrifug
5. column into a new 2 ml collection tube supplied and wash by adding 500 ul HB Buffer Centrifuge at 10 000 x g for 30 seconds Discard the flow through and collection tube 10 11 12 13 Place column into the same 2 ml collecting tube supplied and wash by adding 700 ul DNA Wash Buffer diluted with absolute ethanol Centrifuge 10 000 x g 1 min as above Discard flow through 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 and page 4 If refrigerated the diluted DNA wash buffer must be brought to room temperature before use Repeat step 9 with a second 700 ul DNA Wash Buffer diluted with ethanol Discard liquid and re insert the column to the empty collecting tube centrifuge the column at 15 000 xg for 2 min at room temperature This step is critical in removing traces of ethanol that will interfere with downstream applications Place column into aclean 1 5 ml microfuge tube not suplied To elute DNA add 50 100 ul of Elution Buffer or 10 mM Tris buffer pH 9 0 preheated to 60 C 70 C directly onto the HiBind matrix Allow to soak for 2 min at room temperature Centrifuge at 10 000 x gfor 1 min to Elute DNA Repeat elution step with a second 50 100 ul Elution Buffer Typically a total of 5 15 ug DNA with absorbance ratio Azso Az80 Of 1 7 1 9 can be obtained from 1 gram soil sample Yields vary
6. 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 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 A A ratio Values of 1 7 1 9 generally indicate 85 90 purity The concentration of DNA eluted can be determined as follows Concentration 50 ug ml x Absorbance x Dilution Factor Troubleshooting Guide Problem Possible Cause Suggestions Clogged Incomplete lysis Increase incubation time with Buffer ML1 Column 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 Pulverize material as indicated in liquid homogenization nitrogen to obtain a fine powder Low DNA Clogged column See above yield Poor elution Repeat elution or increase elution volume Incubate the column at 70 C for 5 min before spin Poor binding to Follow protocol closely when adjusting column binding conditions Improper washing DNA Wash Buffer Concentrate must be diluted with ethanol before use Low Extended Resin from the column may be present in Ay60 Azeo centrifugation duri
7. e 10 000 x g for 2 min at room temperature Carefully transfer the upper aqueous phase to aclean 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 another of 200 ul of ML1 Buffer and vortex to mix Centrifuge as above and transfer the upper aqueous phase to tube OPTIONAL Add 5 ul assuming a sample size of 30 mg RNase A and incubate at room temperature for 10 30 minutes Proceed with the protocol Measure the volume of supernatant from step 4 and add one volume of Buffer MBL and vortex at maxi speed for 15s Incubate at 70 C for 10 minutes Add 0 5 volume of absolute ethanol room temperature 96 100 and mix well by vortexing at maxi speed for 15s Tips 300 ul upper aqueous solution add 300 ul Buffer MBL and 300 ul of absolute ethanol 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 collecting tube supplied Centrifuge at 10 000 x g for 1 min at room temperature Discard flow through liquid and re use collection tube Place HiBind 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 Place the
8. ng eluate Avoid centrifugation at speeds higher ratio elution step 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 ML1 An overnight incubation may be necessary Trace protein Following step 8 wash column witha contaminants remain mixture of 300 ul Buffer ML2 300 ul ethanol before proceeding to step 9 No DNA Poor cell lysis Increase incubation time with Buffer ML1 eluted An overnight incubation may be necessary 8 Problem Possible Cause Suggestions 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 MBL and absolute ethanol No ethanol added to DNA Wash Buffer Concentrate Dilute Wash Buffer with the indicated volume of absolute ethanol before first use
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