Kit Manual
Contents
1. Contents IntroductiOn ito Dd eee gels setebaestessoleneehicesetdeleeetlsnaciey 2 storage and Stability eee mette eset A 2 Binding Capacity iai ett eim ete disinterest 2 Kit Contents ue ERI ene eR tene eps 3 Before Starting eee ere prie tiae mtd tette 3 EZgene Mollusc Arthropod DNA Protocol 4 Determination of DNA Quality and Quantity sess 7 Trouble Shooting Guide eene 8 Biomiga EZgene Mollusc gDNA Miniprep Kit Page 1 Introduction The EZgene Mollusc gDNA Kit is designed to extract 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 Samples are homogenized and lysed in a high salt buffer and extracted with chloroform to remove mucopolysaccharides Following a rapid alcohol precipitation step binding conditions are adjusted and DNA further purified using ezBind DNA spin columns While proteins and other contaminants are removed by wash buffer high quality genomic DNA is eluted with elution buffer or sterile water The purified genomic DNA is suitable for downstream applications such as Southern Blot restriction digestion and PCR Storage and Stability All components of the2. EZgene Mollusc gDNA 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 20 C Under 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 All EZgene Mollusc gDNA Kit components are guaranteed for at least 12 months from the date of purchase when stored at 22 C 25 C Binding Capacity Each ezBind DNA column can bind approximately 100 ug DNA Use less than 30 mg of sample per column Page 2 Biomiga EZgene Mollusc gDNA Miniprep Kit Kit content Product GD2414 00 GD2414 01 GD2414 02 ezBind DNA Columns 50 250 2 mL collecting tubes 100 500 Buffer MTL 20 mL 100 mL Buffer MBL 20 mL 100 mL Buffer KB 28 mL 135 mL Proteinase K 30 mg 5 x 30 mg RNase A 20 mg mL 270 uL 1350 uL DNA Wash Buffer 15 mL 3x24mL Elution Buffer 15 mL 70 mL User Manual 1 1 Before Starting Please read the entire booklet to become familiar with the EZgene protocol Dilute DNA Wash Buffer with 100 ethanol as follows GD2414 00 Add 8 mL absolute 96 100 ethanol GD2414 01 Add 60 mL 96 100 ethanol to each bottle GD2414 02 Add 96 mL 96 100 ethanol to each bottle R
3. before proceeding to step 9 F Increase incubation time with Buffer MTL An Poor cell lysis E overnight incubation may be necessary Incomplete Pulverize starting material as indicated in liquid homogenization nitrogen to obtain a fine powder Trace protein contaminants remain Absolute ethanol not added before adding sample to column No ethanol added to DNA Wash Buffer Concentrate Before applying DNA sample to column add Buffer MBL and absolute ethanol Dilute Wash Buffer with the indicated volume of absolute ethanol before first use Page 8 Biomiga EZgene Mollusc gDNA Miniprep Kit
4. econstitute Proteinase K stock solution Vortex vial briefly prior to use We recommend that you aliquot and store vials of reconstituted protease at 20 C GD2414 00 Add 110 uL Elution Buffer to the vial GD2414 01 Add 1 3 mL Elution Buffer to the vial GD2414 02 Add 5 x 1 3 mL Elution Buffer to each vial Biomiga EZgene Mollusc gDNA Miniprep Kit Page 3 EZgene Mollusc Arthropod DNA Protocol Materials to be provided by user fe Microcentrifuge capable of at least 14 000 x g fe ko Nuclease free 1 5 mL or 2 mL microfuge tubes Water bath equilibrated to 65 C Equilibrate sterile ddH O or 10 mM Tris pH 8 5 at 65 C Absolute 96 100 ethanol Chloroform isoamyl alcohol 24 1 fe ko fe ko fe KS 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 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 DNA column TM as binding capacity 100 u
5. erature 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 MTL Buffer and vortex to mix Centrifuge as above and transfer the upper aqueous phase to tube Add one volume of Buffer MBL followed by 5 uL RNase A vortex at maxi speed for 15 s Incubate at 70 C for 10 minutes Add one volume of absolute ethanol room temperature and mix well by vortexing at maxi speed for 15 s Note 500 uL upper aqueous solution add 500 uL Buffer MBL and 500 uL of absolute ethanol Apply 750 uL of the mixture from step 5 including any precipitation that may have formed to an ezBind DNA column Centrifuge at 10 000 x g for 1 min at room temperature Discard flow through liquid and re use collection tube 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 Biomiga EZgene Mollusc gDNA Miniprep Kit Page 5 8 Place the column into another a new 2 mL collection tube supplied and wash by adding 500 uL Buffer KB Centrifuge at 10 000 x g for 30 s Discard the flow through and re use collection tube 9 Place c
6. g may be exceeded Meanwhile difficult tissues may require starting with less than 30 mg tissue and doubling all volumes to ensure adequate lysis Molluscs 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 S1015 39 amp 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 Arthropods 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 Page 4 Biomiga EZgene Mollusc gDNA Miniprep Kit ceramic mortar and pestle are not available homogenize the sample in the microfuge tube using a disposable microtube pestle Proceed to step 2 below Add 350 uL Buffer MTL 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 pL chloroform isoamyl alcohol 24 1 and vortex to mix Centrifuge 10 000 x g for 2 min at room temp
7. m 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 pg mL x Absorbance260 x Dilution Factor Biomiga EZgene Mollusc gDNA Miniprep Kit Page 7 Trouble Clogged Column Low DNA yield No DNA eluted Shooting Guide Possible Cause Inda duh Increase incubation time with Buffer MTL Proteinase p y K An overnight incubation may be necessary Do not use greater than recommended amount of Sample too large starting material For larger samples divide into multiple tubes Incomplete Pulverize material as indicated in liquid nitrogen to homogenization obtain a fine powder 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 binding column conditions I hi DNA Wash Buffer Concentrate must be diluted with eee wen ethanol before use Resin from the column may be present in eluate Avoid Extended centrifugation at speeds higher than specified The centrifugation during material can be removed from the eluate by elution step centrifugation it will not interfere with PCR or restriction digests Increase incubation time with Buffer MTL An Poor cell lysis n overnight incubation may be necessary Following step 8 wash column with a mixture of 300 uL Buffer MBL 300 uL ethanol
8. olumn into collection tube from previous step and add 650 pL DNA Wash Buffer diluted with 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 10 Repeat step 9 with a second 650 uL DNA Wash Buffer diluted with ethanol Discard liquid and collection tube And insert the column into a new collecting tube centrifuge the column at 15 000 x g for 2 min at room temperature This step is critical in removing traces of ethanol that will interfere with downstream applications 1 Place column into a clean 1 5 mL microfuge tube not supplied To elute DNA add 50 uL 100 uL of Elution Buffer or 10 mM Tris buffer pH 9 0 preheated to 60 C 70 C 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 pL 100 uL Elution Buffer Tip To increase DNA Yield add Elution buffer and incubate the column at 60 C 70 C for 5 min before elution Page 6 Biomiga EZgene Mollusc gDNA Miniprep Kit Determination of DNA Quality and Quantity Dilute a portion of the eluted material approximately 10 20 fold in ddH O Measure absorbance at 280 n
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