E.Z.N.A.®Insect DNA Kit - Omega Bio-Tek
Contents
1. DNA Mini Column for 2 minutes at maximum speed to dry the column matrix Note It is important to dry the HiBind DNA Mini Column matrix before elution Residual ethanol may interfere with downstream applications Transfer the HiBind DNA Mini Column to a clean 1 5 mL microcentrifuge tube Add 50 100 uL Elution Buffer sterile deionized water or 10 mM Tris pH 9 0 heated to 70 C directly to the center of the column membrane Let sit at room temperature for 2 minutes 27 28 29 10 E Z N A Insect DNA Kit Protocol Centrifuge at maximum speed for 1 minute Note This represents approximately 70 of bound DNA An optional second elution will yield any residual DNA though at a lower concentration Repeat Steps 25 27 for a second elution step Note Any combination of the following steps can be used to help increase DNA yield After adding the Elution Buffer incubate the column for 5 minutes Increase the elution volume Repeat the elution step with fresh Elution Buffer this may increase the yield but decrease the concentration e Repeat the elution step using the eluate from the first elution this may increase yield while maintaining elution volume Store DNA at 20 C Troubleshooting Guide Please use this guide to troubleshoot any problems that may arise For further assistance please contact the technical support staff toll free at 1 800 832 8896 Increase incubation time with CTL Buffer I2. Bind E Z N A and MicroElute are registered trademarks of Omega Bio tek Inc Qiagen QlAvac and Vacman are all trademarks of their respective companies PCR is a patented process of Hoffman La Roche Use of the PCR process requires a license 12
3. E Z N A Insect DNA Kit D0926 00 5 preps DO926 01 50 preps D0926 02 200 preps May 2013 E Z N A Insect DNA Kit Table of Contents Introduction and 041 1 0 4 2 Kit Contents Storage and Stability 3 Preparing Resgents ae 4 Yield and Quality 1101 5 Ins ct DNA PROLOG een 6 Troubleshooting ns 11 0192100019 oca 12 Manual Revision May 2013 Fi OMEGA bio tek Innovations in nucleic acid isolation Introduction and Overview 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 frozen samples or for samples preserved in alcohol or DNE solution Good results also 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 ability of Omega Bio tek s HiBind matrix to isolate high quality DNA Samples are homogenized and lysed in a high salt buffer containing CTAB and extracted with chloroform to remove polysaccharides Following a rapid alcohol precipitation step binding conditions are adjusted and DNA is further purified using HiBind DNA Mini Columns In this way salts proteins and other contaminants are removed to yield high quality ge
4. e for PCR amplification but fresh or frozen samples should be used for Southern analysis 1 Prepare samples using one of the methods below depending on sample type A Insects i Pulverize no more than 50 mg tissue in liquid nitrogen using a mortar and pestle Note If a ceramic mortar and pestle are not available homogenize the sample in the microcentrifuge tube using a disposable microtube pestle Omega Bio tek Cat No SSI 1015 39 amp SSI 1014 39 ii Transfer the powdered tissue to a clean 1 5 mL microcentrifuge tube not provided iii Proceed to Step 2 below E Z N A Insect DNA Kit Protocol B Arthropods and other soft tissue invertebrates and plant samples i Pulverize no more than 30 mg tissue in liquid nitrogen using a mortar and pestle Note If a ceramic mortar and pestle are not available homogenize the sample in the microcentrifuge tube using a disposable microtube pestle Omega Bio tek 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 59887 will help ii Transfer the powdered tissue to a clean 1 5 mL microcentrifuge tube not provided iii Proceed to Step 2 below Note Amount of starting material depends on the 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 proporti
5. lution add 500 uL CBL Buffer and 500 uL 100 ethanol Insert a HiBind DNA Mini Column into a 2 mL Collection Tube Optional Protocol for Column Equilibration 11 12 13 14 Add 100 uL 3M NaOH to the HiBind DNA Mini Column Let sit for 4 minutes Centrifuge at maximum speed for 20 seconds Discard the filtrate and reuse the Collection Tube PWN gt Transfer 750 uL cleared lysate including any precipitates that may have formed from Step 9 by CAREFULLY aspirating it into the HiBind DNA Mini Column Centrifuge at maximum speed for 1 minute Discard the filtrate and reuse the collection tube Repeat Steps 11 13 until all the remaining samples has been transferred to the HiBind DNA Mini Column 13 16 17 18 19 20 21 22 23 24 25 26 E Z N A Insect DNA Kit Protocol Transfer the HiBind DNA Mini Column into a 2 mL Collection Tube Add 500 uL HBC Buffer Note HBC Buffer must be diluted with isopropanol before use Please see Page 4 for instructions Centrifuge at maximum speed for 30 seconds Discard the filtrate and reuse collection tube Add 700 uL DNA Wash Buffer Note DNA Wash Buffer must be diluted with 100 ethanol prior to use Please see Page 4 for instructions Centrifuge at maximum speed for 1 minute Discard the filtrate and reuse the collection tube Repeat Steps 19 21 for a second DNA Wash Buffer wash step Centrifuge the empty HiBind
6. ncomplete Iysis and Proteinase K Solution An overnight incubation may be necessary Do not use more than the recommended Sample too large amount of starting material For larger samples divide into multiple tubes Incomplete Pulverize starting material in liquid nitrogen homogenization as indicated to obtain a fine powder Clogged column See above Poor elution Repeat elution or increase elution volume Poor binding to column Follow protocol closely when adjusting the binding conditions DNA Wash Buffer must be diluted with 100 ethanol See Page 4 for instructions Improper washing HBC Buffer must be diluted with isopropanol See Page 4 for instructions 100 ethanol not Before applying DNA sample to column add added before adding CBL Buffer and 100 ethanol as indicated in sample to column Steps 7 and 9 Page 8 Resin from the column may be present in el uate 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 Increase incubation time with CTL Buffer Poor cell lysis E and Proteinase K Solution Extended centrifugation Low A A during elution step ratio 11 Ordering Information The following components are available for purchase separately Call Toll Free at 1 800 832 8896 Product ATT Elution Buffer 100 mL PDRO48 DNA Wash Buffer 100 mL 25010 RNase A 400 uL AC117 Hi
7. nomic DNA suitable for downstream applications such as endonuclease digestion thermal cycle amplification and hybridization techniques New in this Edition e This manual has been edited for content and redesigned to enhance user readability HB Buffer has been replaced by HBC Buffer Isopropanol is required and supplied by the user e Equilibration Buffer is no longer included with this kit An optional Column Equilibration Protocol has been added to the protocol for your convenience e Equilibration Buffer is replaced with 3M NaOH provided by the user Kit Contents D0926 00 D0926 01 D0926 02 5 w Storage and Stability All of the E Z N A Insect DNA Kit components are guaranteed for at least 12 months from the date of purchase when stored as follows RNase A must be stored at 2 8 C Proteinase K Solution can be stored at room temperature for up to 12 months For long term storage store Proteinase K Solution at 2 8 C All remaining components should be stored at room temperature During shipment or storage in cool ambient conditions precipitates may form in some of the buffers Dissolve such deposits by warming the solution at 65 C and gently shaking Preparing Reagents 1 Dilute DNA Wash Buffer with 100 ethanol as follows and store at room temperature D0926 02 100 mL per bottle 2 Dilute HBC Buffer with isopropanol as follows and store at room temperature Yield and Quality of DNA Determine the ab
8. on In any event use no more than 50 mg tissue per HiBind DNA Mini Column as the DNA binding capacity 100 ug may be exceeded Difficult tissues may require starting with less than 30 mg tissue and doubling all volumes to ensure adequate lysis 2 Add 350 uL CTL Buffer and 25 uL Proteinase K Solution Vortex briefly to mix 3 Incubate at 60 C for 30 minutes or until entire sample is solubilized Note Actual incubation times may vary and depend on the elasticity of tissues Most samples require no more than 4 hours Alternatively an overnight incubation at 37 C will produce adequate results 4 Add 350 uL chloroform isoamyl alcohol 24 1 Vortex to mix thoroughly 5 Centrifuge at 10 000 x g for 2 minutes at room temperature 10 E Z N A Insect DNA Kit Protocol Carefully transfer the upper aqueous phase to a clean 1 5 mL microcentrifuge tube not provided 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 little upper aqueous phase is present after centrifugation add 200 uL CTL1 Buffer Vortex to mix thoroughly Repeat Steps 5 6 above Add one volume CBL Buffer and 2 uL RNase A Vortex at maximum speed for 15 seconds Incubate at 70 C for 10 minutes Add one volume 100 ethanol Vortex at maximum speed for 15 seconds Note For example for 500 uL upper aqueous so
9. sorbance of an appropriate dilution 20 to 50 fold ofthe sample at 260 nm and then at 280 nm The DNA concentration is calculated as follows DNA concentration Absorbance 260 x 50 x Dilution Factor pg mL A ratio greater than 1 8 indicates greater than 90 nucleic acid Alternatively quantity as well as quality sometimes can be determined best by agarose gel ethidium bromide electrophoresis by comparison to DNA samples of known concentrations Typically the majority of the DNA eluted is in monomeric supercoil form though concatemers may also be present E Z N A Insect DNA Kit Protocol E Z N A Insect DNA Kit Protocol Materials and Equipment to be Supplied by User Microcentrifuge capable of at least 14 000 x g e Nuclease free 1 5 mL or 2 mL microcentrifuge tubes Water baths capable of 70 C 100 ethanol lsopropanol Chloroform Isoamyl alcohol e Optional 3M NaOH Optional Sterile deionized water or 10 mM Tris pH 9 0 Before Starting Prepare DNA Wash Buffer and HBC Buffer according to the instructions in the Preparing Reagents section on Page 4 Prepare a 24 1 solution of chloroform isoamyl alcohol Heat water baths to 60 C and 70 C Heat Elution Buffer to 70 C Insect samples preserved in formalin should be rinsed in xylene and ethanol before processing Results obtained with formalin fixed tissues generally depend on age and size of specimen Purified material is usually adequat
Download Pdf Manuals
Related Search