E-Z 96 Plant DNA Kit
Contents
1. all the samples are completely suspended and that there are no clumps in the solution Clumps will result in low yields 10 11 12 13 E Z 96 Plant DNA Kit Protocols Incubate at 65 C for 10 minutes Mix samples twice during incubation by briefly shaking the plate side to side Centrifuge briefly to collect any drops of liquid from the caps Do not prolong this step Remove and discard the caps Add 140 uL SP2 Buffer to each lysate Seal the wells with new Caps for Round well Plate Vortex at maximum speed or shake the plate side to side vigorously for 20 seconds Centrifuge briefly to collect any drops of liquid from the caps Do not prolong this step Note Do not prolong this step The brief centrifugation prevents the precipitates from freezing to the caps making it difficult to open the caps after incubation at 20 C in next step Incubate at 20 C for 10 minutes Centrifuge at 3 000 6 000 x g for 10 minutes Note Optional E Z 96 Lysate Clearance Plates Cat FL9601 are available for use with this kit at this step Briefly place an E Z 96 Lysate Clearance Plate on top of the 96 well Racked Microtubes provided Transfer the desired volume of supernatant following the 20 C incubation in Step 10 to the E Z 96 Lysate Clearance Plate Centrifuge at 3 000 5 000 x g for 5 minutes Proceed to Step 13 Remove and discard the caps Transfer 400 uL supernatant to the 96 well Racked Microtubes If less than 42. must be diluted with 100 ethanol prior to use Please see Page 5 for instructions 24 25 26 27 28 29 30 31 32 33 34 35 E Z 96 Plant DNA Kit Protocols Centrifuge at 3 000 5 000 x g for 5 minutes Discard the filtrate and reuse the 96 well Square well Plate Repeat Steps 23 25 for a second SPW Wash Buffer wash step Centrifuge at 3 000 5 000 x g for 15 minutes to dry the plate Note It is important to dry the plate membrane before elution Residual ethanol may interfere with downstream applications Remove the AeraSeal Film Transfer the E Z 96 DNA Plate to new 96 well Racked Microtubes provided or a 96 well microplate not provided Add 100 uL Elution Buffer heated at 65 C to each well Seal the plate with new AeraSeal Film Incubate at 65 C for 5 minutes Centrifuge at 5 000 x g for 5 minutes Repeat Steps 30 32 for a second elution step Note To maintain higher DNA concentration second elution may be performed with first eluate Seal the 96 well Racked Microtubes with Caps for Racked Microtubes Store DNA at 20 C 13 E Z 96 Plant DNA Kit Protocols E Z 96 Plant DNA Kit Vacuum Protocol The following protocol is based on using Omega Bio tek s vacuum manifold Cat VAC 03 Materials and Equipment to be Supplied by User Vacuum manifold and vacuum source Centrifuge equipped with swing bucket rotor capable of at least 3 000 x g Water bath oven or incuba
3. vacuum Continue to apply the vacuum for 10 minutes after all liquid has passed through the E Z 96 DNA Plate Turn off the vacuum Discard the filtrate and collection plate Place the E Z 96 DNA Plate upside down on a stack of paper towels and tap several times to remove any residual ethanol Note It is very important to completely dry the E Z 96 DNA Plate before elution If a swing bucket centrifuge with a 96 well plate adaptor is available centrifuge at 5 000 x g for 5 minutes to dry the plate Or if an oven incubator is available dry the plate at 70 C for 10 minutes Place the 96 well Racked Microtubes inside the base of the manifold 17 33 34 35 36 37 E Z 96 Plant DNA Kit Protocols Place the E Z 96 DNA Plate on top of the manifold Add 100 uL Elution Buffer heated to 65 C to each well Let sit at room temperature for 5 minutes Turn on the vacuum source to draw the Elution Buffer through the plate Turn off the vacuum Optional Repeat Steps 34 37 for a second elution step 38 39 18 Note 100 uL Elution Buffer is sufficient to elute up to 85 of the DNA from each well of the E Z 96 DNA Plate A second elution step with same 100 uL elute containing DNA reheated to 65 C will increase yield by up to 10 15 Total DNA yields vary depending on type and quantity of sample Seal the 96 well Racked Microtubes with Caps for Racked Microtubes provided Store DNA at 20 C Troubles
4. 00 uL supernatant is recovered adjust the volume of SP3 Buffer in Step 14 11 14 15 16 17 E Z 96 Plant DNA Kit Protocols Add 1 5 volumes SP3 Buffer A precipitate may form at this point it will not interfere with DNA isolation Note For example if 400 uL lysate was transferred add 600 uL SP3 Buffer Seal the microtubes with Caps for Racked Microtubes provided Vortex at maximum speed or shake the plate side to side vigorously for 20 seconds Centrifuge briefly to collect any drops of liquid from the caps Do not prolong this step Place the E Z 96 DNA Plate on to a 96 well Square well Plate provided Optional Protocol for Plate Equilibration 18 19 20 21 22 23 12 Add 150 uL 3M NaOH to each well Let sit at room temperature for 4 minutes Centrifuge at 3 000 5 000 x g for 2 minutes Discard the filtrate and reuse the 96 well Square well Plate A WN Carefully transfer 1 mL sample to the E Z 96 DNA Plate Be careful not to spill sample liquid onto the rims of the wells during the transfer Seal the E Z 96 DNA Plate with AeraSeal Film provided Centrifuge at 3 000 5 000 x g for 5 minutes or until all the sample has passed through the HiBind membrane Discard the filtrate and reuse the 96 well Square well Plate Remove the AeraSeal Film Add 800 uL SPW Wash Buffer to each well of the E Z 96 DNA Plate Seal the plate with new AeraSeal Film Note SPW Wash Buffer
5. E Z 96 Plant DNA Kit D1086 01 1 x 96 preps D1086 02 4 x 96 preps D1086 03 20 x 96 preps April 2013 E Z 96 Plant DNA Kit Table of Contents la 1e 10 ao A ee 2 Illustrated Prototols insano 3 Kit Contents Storage and Stability 4 Preparing Reagents Cleaning Plates 5 Guidelines for Vacuum Manifold 6 Disruption of Plant TISSUCS iii 8 E Z 96 Plant DNA Centrifugation Protocol 10 E Z 96 Plant DNA Vacuum Protocol 14 Troubleshooting Gulden 19 CPLR etnia iii iia 20 Manual Revision April 2013 Fi OMEGA bio tek Innovations in nucleic acid isolation Introduction E Z 96 Plant DNA Kit allows for the rapid and reliable isolation of high quality total cellular DNA from a wide variety of plant species and tissues in a 96 well plate format E Z 96 Plant DNA Kit adapted the buffer system from Omega Bio tek s SP Plant DNA system which is used to process a variety of plant types particularly those with unusually high levels of phenolic compounds or polysaccharides such as cotton pine and peanut samples Up to 50 mg wet tissue or 12 mg dry tissue can be processed in each well in less than one hour The system combines the reversible nucleic acid binding properties of the HiBind matrix with the speed and versatility of the E Z 96 DNA Plate to eliminate polysaccharides phenolic compounds and enzyme inhibitors from plant tissue lysates Purified DNA is suitable for PCR restriction digestion an
6. NE ecseiin AICA u 96 well Square well Plates 2 2 mL are reusable see Page 5 for cleaning instructions Storage and Stability All components of the E Z 96 Plant DNA Kit are guaranteed for at least 12 months from date of purchase when stored as follows Store RNase A at 2 8 C All other components should be stored at room temperature During shipment or storage in cool ambient conditions precipitates may form in SP1 Buffer and SP3 Buffer Dissolve such deposits by warming the solution at 37 C and gently shaking Preparing Reagents Dilute SP3 Buffer with 100 ethanol as follows and store at room temperature D1086 02 200 mL D1086 03 500 mL per bottle Dilute SPW Wash Buffer with 100 ethanol as follows and store at room temperature D1086 01 160 mL D1086 02 640 mL D1086 03 800 mL per bottle Prepare SP1 Buffer Add 2 uL RNase A per 400 uL SP1 Buffer Cleaning Instructions for 96 well Square well Plates 96 well Square well Plates can be used to collect filtrate from the E Z 96 DNA Plate They are designed for repeated use Wash the plates thoroughly in tap water after each use Let sit for 5 minutes at room temperature in 0 5M HCI Rinse with distilled water Used plates can also be autoclaved after washing Guidelines for Vacuum Manifold The following is required for use with the Vacuum Protocol A Vacuum Manifold We recommend Omega Bio tek s VAC 03 Other Compatible Vacuum Manifolds Qiagen QlAv
7. Plant DNA Kit Protocol Centrifugation Protocol Materials and Equipment to be Supplied by User Centrifuge equipped with swing bucket rotor capable of at least 3 000 x g Water baths ovens or incubators capable of 80 C Vortexer 100 ethanol Liquid nitrogen for freezing disrupting samples for fresh frozen specimens Equipment for disrupting plant tissue Ice freezer or 96 well cryorack at 20 C Optional 3M NaOH Before Starting 10 Prepare SP1 Buffer SP3 Buffer and SPW Wash Buffer according to Preparing Reagents section on Page 5 Set a water bath oven or incubator to 65 C Heat SP1 Buffer and Elution Buffer to 65 C SP1 Buffer should be equilibrated to 80 C if liquid nitrogen is used for sample disruption Homogenize plant tissue following one of the methods described in the Disruption of Plant Tissue section on Pages 8 9 Transfer up to 15 mg dry powdered tissue or 50 mg fresh or frozen tissue to a 96 well Round well Plate provided Note No more than 50 mg wet weight or 15 mg dry weight starting material is recommended More or less can be used depending on results Water content and buffer absorption of samples affect optimal starting amounts Add 400 uL SP1 Buffer to each sample Seal the wells with Caps for Round well Plate provided Vortex to mix thoroughly Note SP1 Buffer must be mixed with RNase A before use Please see the Preparing Reagents section on Page 5 for instructions Ensure that
8. ac24 Sigma AldrichVM20 Promega Vacman or manifold with standard Luer connector B Vacuum Flask C Vacuum Tubing D Vacuum Source review tables below for pressure settings Recommended Pressure mbar VAC 03 200 to 400 Conversion from millibars Multiply by Millimeters of Mercury mmHg 0 75 Kilopascals kPa 0 1 Inches of Mercury inchHg 0 0295 Tors Tor Atmospheres atmos 0 000987 Pounds per Square Inch psi 0 0145 Illustrated Vacuum Setup De LS Omega Bio tek s VAC 03 C Vacuum Tubing D Vacuum Source A Vacuum Manifold B Vacuum Flask Guidelines for Vacuum Manifold DNA Bind and Wash Setup E Z 96 DNA Plate Vacuum Manifold Top Waste Collection Vacuum Manifold Base Standard Elution Setup Optional Elution Setup E Z 96 DNA Plate E Z 96 DNA Plate Vacuum Manifold Top Vacuum Manifold Top Microplate 300 uL Racked Microtubes Racked Microtubes Microplate 300 uL Vacuum Manifold Vacuum Manifold E Sie Base Se Base 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 dry 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 Disposab
9. d hybridization applications If using the E Z 96 Plant DNA Kit for the first time please read this booklet to become familiar with the procedures Dry or fresh plant tissue is disrupted and lysed in a specially formulated buffer containing a proprietary detergent mixture Proteins polysaccharides and cellular debris are precipitated Binding conditions are adjusted and the sample is transferred to the E Z 96 DNA Plate Two rapid wash steps remove trace contaminants such as residual polysaccharides and pure DNA is eluted in water or low ionic strength buffer Purified DNA can be used directly in downstream applications without the need for further purification Optional E Z 96 Lysate Clearance Plates Product No FL96 01 are available for use with this kit New In this Edition 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 Centrifugation Protocol Vacuum Protocol Collect Plant Tissue and Homogenize Collect Plant Tissue and Homogenize Lyse and Precipitate Polysaccharides Lyse and Precipitate Polysaccharides Transfer Cleared Lysate and Adjust Binding Conditions Transfer Cleared Lysate and Adjust Binding Conditions Bind and Wash 2X Bind and Wash 2X Vacuum Dry Membrane Dry Membrane Elute Elute Kit Contents TIEN
10. fer 25 mL RNase A 400 uL Sealing Film AeraSeal Film 96 well Square well Plate 2 2 mL 96 well Round well Plate 1 2 mL RNase A 400 uL E Z 96 Homogenizer Plates 4 x 96 E Z 96 Lysate Clearance Plates 10 x 96 Vacuum Manifold VAC 03 HiBind E Z N A and MicroElute are registered trademarks of Omega Bio tek Inc Qiagen QlAvac and Vacman are all trademarks of their respected companies PCR is a patented process of Hoffman La Roche Use of the PCR process requires a license
11. for 10 minutes Mix samples twice during incubation by briefly shaking the plate side to side Centrifuge briefly to collect any drops of liquid from the caps Do not prolong this step Remove and discard the caps Add 140 uL SP2 Buffer to each lysate Seal the wells with new Caps for Round well Plate Vortex at maximum speed or shake the plate side to side vigorously for 20 seconds Centrifuge briefly to collect any drops of liquid from the caps Do not prolong this step Note Do not prolong this step The brief centrifugation prevents the precipitates from freezing to the caps making it difficult to open the caps after incubation at 20 C in next step Incubate at 20 C for 10 minutes Centrifuge at 3 000 6 000 x g for 10 minutes Note Optional E Z 96 Lysate Clearance Plates Cat FL9601 are available for use with this kit at this step Briefly place an E Z 96 Lysate Clearance Plate on top of the 96 well Racked Microtubes provided Transfer the desired volume of supernatant following the 20 C incubation in Step 10 to the E Z 96 Lysate Clearance Plate Centrifuge at 3 000 5 000 x g for 5 minutes Proceed to Step 13 15 12 13 14 15 16 E Z 96 Plant DNA Kit Protocols Remove and discard the caps Transfer 400 uL supernatant to the 96 well Racked Microtubes If less than 400 uL supernatant is recovered adjust the volume of SP3 Buffer in Step 14 Add 1 5 volumes SP3 Buffer A precipitate may form a
12. hooting 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 Possible Problems and Suggestions Following precipitation with SP2 Buffer Debris carryover make sure no particulate material is transferred F Do not exceed suggested amount of Clogged well Sample too viscous Sugg starting material Incomplete precipitation following addition of SP2 Buffer Problem Cause Solution Incomplete disruption of mp rup Completely homogenize sample starting material Decrease the amount of starting material Increase RCF or time of centrifugation after addition of SP2 Buffer Poor lysis of tissue or increase the amount of SP1 Buffer and SP2 Buffer Low DNA yield Increase elution volume to 200 uL and DNA remains bound to incubate the plate at 65 C for 5 minutes column before centrifugation Dilute SPW Wash Buffer by adding DNA washed off appropriate volume of 100 ethanol prior to use Page 5 Repeat wash step with SPW Wash Buffer Problems in downstream Following the second wash spin ensure applications Ethanol carryover that the plate is completely dried before elution 19 Ordering Information The following components are available for purchase separately Call Toll free at 1 800 832 8896 SP1 Buffer 250 mL SP2 Buffer 60 mL SP3 Buffer 100 mL Elution Buffer 100 mL SPW Wash Buf
13. le 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 50 mg for first time users It is very important to not overload the E Z 96 DNA Plate 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 50 mg tissue If results obtained are satisfactory then increase amount of starting material Best results are obtained with young leaves or needles Although various means of sample disruption can be used for this kit such as beads or pestles we recommend grinding the sample in liquid nitrogen 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 OBI Cat SSI 1015 39 Alternatively allow the liquid nitrogen to evaporate and store the samples at 70 C for later use For critical wo
14. rk 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 96 well racked microtubes or 96 well deep well plate Note Do not allow the sample to thaw during handling and weighing To prevent the sample from thawing keep the rack or plate 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 one 3 4 mm stainless steel bead to each well of a 96 well round well plate 2 Close the individual tubes with Cap Strips 3 Freeze the sample in liquid nitrogen Note Lyophilized Dried samples do not require freezing with liquid nitrogen 4 Place the racks or plates into the clamps of the homogenizer 5 _Homogenize for 60 90 seconds at 30 Hz Tissue samples are disrupted and simultaneously homogenized with the shearing and crushing action of the beads Refer to manufacturer s protocol regarding use of liquid nitrogen with the homogenizer E Z 96 Plant DNA Kit Protocols E Z 96
15. t this point it will not interfere with DNA isolation Note For example if 400 uL lysate was transferred add 600 uL SP3 Buffer Prepare the vacuum manifold according to manufacturer s instructions Place an E Z 96 DNA Plate on the top part of the vacuum manifold Place the waste collection tray inside the base of the manifold Seal any unused wells with sealing film not provided Optional Protocol for Plate Equilibration 17 18 19 20 16 Add 150 uL 3M NaOH to each well Let sit at room temperature for 4 minutes Turn on the vacuum source to draw the NaOH through the column Turn off the vacuum B WN Transfer 1 mL sample including any precipitate that may have formed from Step 14 to the E Z 96 DNA Plate Turn on the vacuum source to draw the sample through the plate Turn off the vacuum Repeat Steps 17 19 until all the sample has been transferred to the E Z 96 DNA Plate 21 22 23 24 25 26 27 28 29 30 31 32 E Z 96 Plant DNA Kit Protocols Add 800 uL SPW Wash Buffer to each well Note SPW Wash Buffer must be diluted with 100 ethanol prior to use Please see Page 5 for instructions Turn on the vacuum source to draw the SPW Wash Buffer through the plate Turn off the vacuum Repeat Steps 21 23 for a second SPW Wash Buffer wash step Add 400 uL 100 ethanol to each well Turn on the vacuum source to draw the ethanol through the plate Turn off the
16. tor capable of 80 C Vortexer 100 ethanol Liquid nitrogen for freezing disrupting samples For Fresh Frozen Specimens Equipment for disrupting plant tissue Ice freezer or 96 well cryorack at 20 C Sealing film Optional 3M NaOH Before Starting 14 Prepare SP1 Buffer SP3 Buffer and SPW Wash Buffer according to Preparing Reagents section on Page 5 Set a water bath oven or incubator to 65 C Heat SP1 Buffer and Elution Buffer to 65 C SP1 Buffer should be equilibrated to 80 C if liquid nitrogen is used for sample disruption Homogenize plant tissue following one of the methods described in the Disruption of Plant Tissue section on Pages 8 9 Transfer up to 15 mg dry powdered tissue or 50 mg fresh or frozen tissue to a 96 well Round well Plate provided Note No more than 50 mg wet weight or 15 mg dry weight starting material is recommended More or less can be used depending on results Water content and buffer absorption of samples affect optimal starting amounts 10 11 E Z 96 Plant DNA Kit Protocols Add 400 uL SP1 Buffer to each sample Seal the wells with Caps for Round well Plate provided Vortex to mix thoroughly Note SP1 Buffer must be mixed with RNase A before use Please see the Preparing Reagents section on Page 5 for instructions Ensure that all the samples are completely suspended and that there are no clumps in the solution Clumps will result in low yields Incubate at 65 C
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