E.Z.N.A.®Plant DNA DS Mini Kit
Contents
1. Mini Columns 200 Homogenizer Mini Columns 200 Elution Buffer 100 mL DNA Wash Buffer 100 mL RNase A 400 uL Part Number SSI 1210 00 SSI 1310 00 DNACOL 02 HCR003 PDR048 PS010 AC117 HiBind 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 PCRis a patented process of Hoffman La Roche Use of the PCR process requires a license 11 Notes2. technical support staff toll free at 1 800 832 8896 Following transfer from Step 7 cell debris may have transferred Carryover of debris Clogged column Do not exceed suggested amount of starting material For both dry and fresh samples obtain a fine homogeneous powder before adding CSPL Buffer Sample too viscous Incomplete disruption of starting material Decrease amount of starting material Poor Iysis of sample or increase amount of CSPL Buffer and Proteinase K Solution Increase elution volume to 200 uL and incu bate on column at 65 C for 5 minutes before centrifugation Dilute DNA Wash Buffer by adding DNA washed off appropriate volume of 100 ethanol prior to use DNA remains bound to column If you are unable to transfer 550 uL after Difficulty transferring lysis with CSPL Buffer increase the buffer sample after lysis amount so that 550 uL can be successfully transferred Problem Solution DNA Wash Buffer must be at room Salt carryover Problems in temperature downstream Ran Following the second wash spin ensure applications Ethanol carryover that the column is dried by centrifuging 2 minutes at maximum speed 10 Ordering Information The following components are available for purchase separately Call Toll Free at 1 800 832 8896 DNase RNase free microcentrifuge tubes 1 5 mL 500 pk 10 pk cs DNase RNase free microcentrifuge tubes 2 0 mL 500 pk 10 pk cs HiBind DNA
3. 500 uL HBC Buffer Note HBC Buffer must be diluted with isopropanol before use Please see Page 4 for instructions Centrifuge at 12 000 x g for 1 minute 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 12 000 x g for 1 minute Discard the filtrate and reuse the collection tube 22 23 24 25 26 27 28 29 30 31 E Z N A Plant DNA DS Mini Kit Protocol Repeat Steps 19 21 for a second DNA Wash Buffer wash step Centrifuge the empty HiBind DNA Mini Column for 2 minutes at 12 000 x g 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 not provided Add 50 100 uL Elution Buffer heated to 65 C directly to the center ofthe column membrane Let sit at room temperature for 1 minute Centrifuge at 12 000 x g for 1 minute Transfer the filtrate to the center ofthe HiBind DNA Mini Column membrane Let sit at room temperature for 1 minute Centrifuge at 12 000 x g for 1 minute Store filtrate containing DNA at 20 C Troubleshooting Guide Please use this guide to troubleshoot any problems that may arise For further assistance please contact the
4. Ca OMEGA Innovations in nucleic acid isolation Product Manual E Z N A Plant DNA DS Mini Kit D2411 00 5 preps D2411 01 50 preps May 2015 For research use only Not intended for diagnostic testing E Z N A Plant DNA DS Mini Kit Table of Contents Introduction and OVErVIEW ccssscssscssecneccscccseesscesseeseecsseeees Kit Contents Storage and Stability Preparing Reagan Disruption HOmMogenilzat on Protocol for Fresh Frozen Dry Samples Troubleshooting Guide unse A see esse Manual Revision May 2015 Fi OMEGA bio tek Innovations in nucleic acid isolation Introduction and Overview The E Z N A Plant DNA DS Mini Kit is designed for efficient recovery of genomic DNA up to 30 kb in size from fresh frozen or dried plant tissue samples rich in polysaccharides polyphenols or those having a lower DNA content Up to 50 mg wet tissue can be processed in less than 1 hour The system combines the reversible nucleic acid binding properties of the HiBind matrix with the speed and versatility of spin column technology to eliminate polysaccharides phenolic compounds and enzyme inhibitors from plant tissue lysates Purified DNA is suitable for PCR restriction digestion and hybridization applications This procedure relies on the well established properties of the cationic detergent cetyltrimethyl ammonium bromide CTAB in conjunction with the unique binding system to increase yields and provide hi
5. e samples at 70 C for later use For critical work such as PCR and cloning pestles are best used a single time then soaked in a dilute bleach solution immediately after use until clean Disposable pestles may be autoclaved several times For standard Southern analysis the same pestle can be reused several times to grind multiple tissue samples by rinsing with ethanol and carefully wiping the surfaces clean between samples Transfer the ground sample into a 1 5 mL microcentrifuge tube Note Do not allow the sample to thaw during handling and weighing To prevent the sample from thawing keep the samples on a bed of dry ice Disruption of Plant Tissues Disrupt Samples With Commercial Homogenizers Fresh frozen and dried plant tissue can be effectively disrupted and homogenized by rapid agitation in the presence of beads For Fresh Frozen and Lyophilized Dried Tissue 1 Add two 3 4 mm stainless steel bead or ceramic beads to each vial 2 Close the individual vial 3 Place the racks or plates into the clamps of the homogenizer 4 Homogenize for 60 90 seconds at 30 Hz Tissue samples are disrupted and simultaneously homogenized with the shearing and crushing action of the beads E Z N A Plant DNA DS Mini Kit E Z N A Plant DNA DS Mini Kit Wet Frozen Dry Tissue Protocol Materials and Equipment to be Supplied by User Microcentrifuge capable of at least 12 000 x g Waterbath capable of 65 C Vortexer Nucl
6. ease free 1 5 and 2 mL microcentrifuge tubes 100 isopropanol 100 ethanol Sample Disruption Method See Pages 5 6 Before Starting Prepare the DNA Wash Buffer and HBC Buffer according to the instructions on Page 4 Heat the Elution Buffer to 65 C 1 Prepare 10 50 mg wet frozen tissue or 2 10 mg dry tissue in a 1 5 or 2 mL microcentrifuge tube vial not provided according to Pages 5 6 For best results use a commercial homogenizer if available 2 Add 700 uL CSPL Buffer and 20 uL Proteinase K Solution Vortex vigorously to mix Make sure to disperse all clumps 3 Incubate at 65 C for 30 minutes 4 Centrifuge at 12 000 x g for 3 minutes 5 Insert aHomogenizer Mini Column into a 2 mL Collection Tube 6 Transfer 550 uL cleared supernatant to the Homogenizer Mini Column 7 Centrifuge at 12 000 x g for 1 minute 10 11 12 13 14 15 16 17 18 19 20 21 E Z N A Plant DNA DS Mini Kit Protocol Transfer the filtrate to anew 2 mL microcentrifuge tube not provided Add 5 uL RNase A Let sit at room temperature for 5 minutes Add 525 uL RBB Buffer and 525 uL XP2 Buffer Vortex to mix thoroughly Insert a HiBind DNA Mini Column into a2 mL Collection Tube Transfer 750 uL lysate from Step 9 to the HiBind DNA Mini Column Centrifuge at 12 000 x g for 1 minute Discard the filtrate and reuse the collection tube Repeat Steps 12 14 to transfer the remaining lysate Add
7. gh quality DNA The system eliminates the need for chloroform extractions traditionally associated with CTAB based lysis methods Samples are homogenized and lysed in a high salt buffer containing CTAB binding conditions are adjusted and DNA is purified using a HiBind DNA Mini Column 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 applications Kit Contents aa Tosmnen osas 10 mM Tris HCI pH 8 5 Storage and Stability All of the E Z N A Plant DNA DS Mini 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 All remaining components should be stored at room temperature During shipment or storage in cool ambient conditions precipitates may form in HBC Buffer Dissolve such deposits by warming the solution at 37 C and gently shaking Preparing Reagents 1 Dilute DNA Wash Buffer with 100 ethanol as follows and store at room temperature D2411 01 100 mL 2 Dilute HBC Buffer with 100 isopropanol as follows and store at room temperature Disruption of Plant Tissues 1 Grind samples with pestle A Dry Specimens Drying allows storage of field specimens for prolonged periods of time prior to processing Samples can be dried overnight in a 45 C oven powdered and stored dr
8. y at room temperature To prepare dried samples place 15 mg of dried tissues into a microcentrifuge tube 1 5 mL tubes are recommended and grind using a pellet pestle Disposable Kontes pestles work well and are available from Omega Bio tek Cat SSI 1014 39 amp SSI 1015 39 For critical work such as PCR and cloning pestles are best used a single time then soaked in a dilute bleach solution immediately after use until clean Disposable pestles may be autoclaved several times A fine powder will ensure optimal DNA extraction and yield Fresh Frozen Specimens Due to the tremendous variation in water and polysaccharide content of plants sample size should be limited to 30 mg for first time users It is very important to not overload the HiBind DNA Mini Column Too much starting material will decrease the yield and purity due to inefficient lysis However for some plant species increasing the starting material can increase DNA yield We recommend starting with 30 mg tissue If results obtained are satisfactory then increase amount of starting material Best results are obtained with young leaves or needles To prepare samples collect tissue in a 1 5 mL or 2 mL microcentrifuge tube and dip the tube in liquid nitrogen with a pair of tweezers to fill the tube Grind the tissue using disposable Kontes pellet pestles which are available from Omega Bio tek Cat SSI 1015 39 Alternatively allow the liquid nitrogen to evaporate and store th
Download Pdf Manuals
Related Search