E.Z.N.A.®SP Plant DNA Maxi Kit - Omega Bio-Tek
Contents
1. size should be limited to 1 g for first time users It is very important to not overload the HiBind DNA Maxi 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 1 g 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 30 mL mortar and freeze by adding liquid nitrogen Grind the tissue using a clean pestle Alternatively allow the liquid nitrogen to evaporate and store the 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 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 50 mL centrifuge tube Note Do not allow the sample to thaw during handling and weighing To prevent the sample from thawing keep the tubes on a bed of dry ice E Z N A SP Plant DNA Maxi Kit Protocols E Z N A SP Plant DNA Maxi Kit2. Ca OMEGA Innovations in nucleic acid isolation Product Manual E Z N A SP Plant DNA Maxi Kit D5538 00 2 preps D5538 01 5 preps February 2014 For research use only Not intended for diagnostic testing E Z N A SP Plant DNA Maxi Kit Table of Contents Introduction and OVEFVIEW csccsesssscsecssecseecseecseecseecneceseersees 2 Kit Contents Storage and Stability sssssssssssesssssssssssssssssssee 3 Preparing Reagents ssseesssserseseeessseresseeessseesseseossssossssossssesssss 4 Sample Preparation ccscssssssssssseesseesecsscsssesssseessseseesesencensessees 5 SP Plant DNA Maxi Protocol Dried SampleS sssssssssssss 6 SP Plant DNA Maxi Protocol Fresh Frozen Samples 10 Troubleshooting Guide ssssssssseerssssssssssseeessessssssssreeseessesssss 14 Oke Va To PREE EE E EEA 15 Manual Revision February 2014 N OMEGA bio tek Innovations in nucleic acid isolation Introduction and Overview The E Z N A SP Plant DNA Maxi Kit is specially designed for rapid and reliable isolation of high quality total cellular DNA from plant species containing high levels of phenolic compounds and polysaccharides Up to 1 g wet tissue or 250 mg dry 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 inh
3. Let sit on ice for 10 minutes E Z N A SP Plant DNA Maxi Kit Protocols 6 Centrifuge at maximum speed 3 000 x g for 6 minutes at room temperature Note Some plant materials can generate very viscous lysates and large amounts of precipitates during this step The preparation of a cleared lysate is essential to prevent clogging of the HiBind DNA Maxi Column For some plant samples it is difficult to clear the lysate via a single centrifugation step because not all particulate matter forms a compact pellet Omega Homogenizer Maxi Columns can efficiently remove most cell debris and precipitates in next step 7 Carefully transfer the supernatant to a Homogenizer Maxi Column Do not disturb the pellet 8 Centrifuge at maximum speed for 5 minutes Note Longer centrifugation does not improve yield The Homogenizer Column will remove most precipitates and cell debris but a small amount might pass through and form a pellet in the Collection Tube 9 Carefully transfer the cleared lysate to a new 50 mL centrifuge tube Do not disturb the pellet Measure the volume of the lysate for the next step 10 Add 1 5 volumes SP3 Buffer Immediately vortex to obtain a homogeneous mixture If precipitation can be seen at this point break the precipitation by passing through a needle using a syringe or pipetting up and down 10 15 times Important Reserve the lysate for later use Do not use in Step 11 Note SP3 Buffer must be diluted with et
4. Protocol Dried Samples Materials and Equipment to be Supplied by User Centrifuge capable of at least 3 000 x g Vortexer Nuclease free 50 mL centrifuge tubes capable of withstanding 3 000 x g Incubator heat block or water bath capable of 65 C 100 ethanol RNase A solution at 50 mg mL e Ice bucket e Optional for fresh samples liquid nitrogen for freezing grinding samples Before Starting Prepare SPW Wash Buffer and SP3 Buffer according to the Preparing Reagents section on Page 4 Set an incubator heat block or water bath to 65 C e Heat Elution Buffer to 65 C Prepare an ice bucket e Prepare an RNase A solution at 50 mg mL 1 Transfer up to 250 mg dried powdered tissue to a 50 mL centrifuge tube 2 Add 7 mL SP1 Buffer and 20 uL RNase A solution 50 mg mL Vortex at maximum speed to mix thoroughly Do not mix SP1 Buffer and RNase A before use Note Ensure that all the samples are completely suspended and that there are no clumps in the solution Clumps will result in low yields 3 Incubate at 65 C for 30 60 minutes Invert the tube several times during incubation to mix 4 Add 2 5 mL SP2 Buffer Vortex to mix thoroughly 5 Let sit on ice for 10 minutes E Z N A SP Plant DNA Maxi Kit Protocols 6 Centrifuge at maximum speed 3 000 x g for 6 minutes at room temperature Note Some plant materials can generate very viscous lysates and large amounts of precipitates during this st
5. ent or storage in cool ambient conditions precipitates may form in SP1 Buffer and or SP3 Buffer Dissolve such deposits by warming the solution at 37 C and gently shaking Preparing Reagents 1 Dilute SP3 Buffer with 100 ethanol as follows and store at room temperature 2 Dilute SPW Wash Buffer with 100 ethanol as follows and store at room temperature D5538 01 100 mL Sample Preparation Choose the most appropriate protocol Procedures are described for both dried and fresh frozen samples 250 mg powdered tissue Recommended Sample Preparation 1 Dried 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 up to 250 mg dried tissue into a 50 mL centrifuge tube and grind using a pellet pestle 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 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 Disposable pestles may be autoclaved several times A fine powder will ensure optimal DNA extraction and yield 2 Fresh Frozen Specimens Due to the tremendous variation in water and polysaccharide content of plants sample
6. ep The preparation of a cleared lysate is essential to prevent clogging of the HiBind DNA Maxi Column For some plant samples it is difficult to clear the lysate via a single centrifugation step because not all particulate matter forms a compact pellet Omega Homogenizer Maxi Columns can efficiently remove most cell debris and precipitates in next step 7 Carefully transfer the supernatant to a Homogenizer Maxi Column Do not disturb the pellet 8 Centrifuge at maximum speed for 5 minutes Note Longer centrifugation does not improve yield The Homogenizer Column will remove most precipitates and cell debris but a small amount might pass through and form a pellet in the Collection Tube 9 Carefully transfer the cleared lysate to a new 50 mL centrifuge tube Do not disturb the pellet Measure the volume of the lysate for the next step 10 Add 1 5 volumes SP3 Buffer Immediately vortex to obtain a homogeneous mixture If precipitation can be seen at this point break the precipitation by passing through a needle using a syringe or pipetting up and down 10 15 times Important Reserve the lysate for later use Do not use in Step 11 Note SP3 Buffer must be diluted with ethanol before use Please see the Preparing Reagents section on Page 4 for instructions Optional Protocol for Column Equilibration Important Do not add 3M NaOH to the lysate from Step 10 Add 3 mL 3M NaOH to the HiBind DNA Maxi Column Let sit at room temperature f
7. gher DNA concentration in the first eluate Alternatively DNA concentration can be increased by using the first eluate for the second elution step 25 Store DNA at 20 C E Z N A SP Plant DNA Maxi Kit Protocols E Z N A SP Plant DNA Maxi Kit Protocol Fresh Frozen Specimens Materials and Equipment to be Supplied by User Centrifuge capable of at least 3 000 x g Vortexer Nuclease free 50 mL centrifuge tubes capable of withstanding 3 000 x g Incubator heat block or water bath capable of 65 C 100 ethanol RNase A solution at 50 mg mL Ice bucket Optional for fresh samples liquid nitrogen and mortar and pestle for freezing and grinding samples Before Starting 10 Prepare SPW Wash Buffer and SP3 Buffer according to the Preparing Reagents section on Page 4 Set an incubator heat block or water bath to 65 C Heat Elution Buffer to 65 C Prepare an ice bucket Prepare an RNase A solution at 50 mg mL Transfer up to 1 g ground plant tissue to a 50 mL centrifuge tube not provided Add 5 mL SP1 Buffer and 20 uL RNase A stock solution 50 mg mL Vortex at maximum speed to mix thoroughly Do not mix SP1 Buffer and RNase A before use Note 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 for 30 minutes Invert the tube several times during incubation to mix Add 1 75 mL SP2 Buffer Vortex to mix thoroughly
8. hanol before use Please see the Preparing Reagents section on Page 4 for instructions Optional Protocol for Column Equilibration Important Do not add 3M NaOH to the lysate from Step 10 Add 3 mL 3M NaOH to the HiBind DNA Maxi Column Let sit at room temperature for 4 minutes Centrifuge at 3 000 x g for 3 minutes Discard the filtrate and reuse the collection tube P oma 11 11 12 13 14 15 16 17 18 19 20 21 22 23 12 E Z N A SP Plant DNA Maxi Kit Protocols Transfer 15 mL lysate including any precipitation from Step 10 to the HiBind DNA Maxi Column Centrifuge at maximum speed for 5 minutes Discard the filtrate and reuse the Collection Tube Repeat Steps 11 13 until all of the lysate has been transferred to the column Add 10 mL SPW Wash Buffer Note SPW Wash Buffer must be diluted with ethanol before use Please see the Preparing Reagents section on Page 4 for instructions Centrifuge at maximum speed for 4 minutes Discard the filtrate and reuse the Collection Tube Repeat Steps 15 17 for a second SPW Wash Buffer wash step Centrifuge the empty column at maximum speed for 10 minutes Note It is important to dry the column membrane before elution Residual ethanol may interfere with downstream applications Transfer the HiBind DNA Maxi Column to a 50 mL centrifuge tube not provided Add 2 mL Elution Buffer heated to 65 C directly to the center of column ma
9. ibitors from plant tissue lysates Purified DNA is suitable for PCR restriction digestion and hybridization techniques If using the E Z N A SP Plant DNA Maxi Kit for the first time please read this booklet to become familiar with the procedures Dry or fresh frozen plant tissue is disrupted and lysed in a specially formulated buffer containing detergent Binding conditions are adjusted and the sample is transferred to a HiBind DNA Maxi Column 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 directly used in downstream applications without the need for further purification New in this Edition 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 Binding Capacity Each HiBind DNA Maxi Column can bind up to 2 mg genomic DNA Using more than 2 g fresh plant tissue is not recommend Kit Contents HiBind DNA Maxi Columns and Homogenizer Columns have been inserted into the 50 mL Collection Tubes for your convenience Storage and Stability All of the E Z N A SP Plant DNA Maxi Kit components are guaranteed for at least 12 months from the date of purchase when stored as follows All components should be stored at room temperature During shipm
10. or 4 minutes Centrifuge at 3 000 x g for 3 minutes Discard the filtrate and reuse the collection tube P oma 11 12 13 14 15 16 17 18 19 20 21 22 23 E Z N A SP Plant DNA Maxi Kit Protocols Transfer 15 mL lysate including any precipitation from Step 10 to the HiBind DNA Maxi Column Centrifuge at maximum speed for 5 minutes Discard the filtrate and reuse the Collection Tube Repeat Steps 11 13 until all of the lysate has been transferred to the column Add 10 mL SPW Wash Buffer Note SPW Wash Buffer must be diluted with ethanol before use Please see the Preparing Reagents section on Page 4 for instructions Centrifuge at maximum speed for 4 minutes Discard the filtrate and reuse the Collection Tube Repeat Steps 15 17 for a second SPW Wash Buffer wash step Centrifuge the empty column at maximum speed for 10 minutes Note It is important to dry the column membrane before elution Residual ethanol may interfere with downstream applications Transfer the HiBind DNA Maxi Column to a 50 mL centrifuge tube not provided Add 2 mL Elution Buffer heated to 65 C directly to the center of column matrix Let sit at room temperature for 5 minutes Centrifuge at maximum speed for 3 minutes E Z N A SP Plant DNA Maxi Kit Protocols 24 Repeat Steps 21 23 for a second elution step Note This step may be performed using another 50 mL centrifuge tube to maintain a hi
11. trix Let sit at room temperature for 5 minutes Centrifuge at maximum speed for 3 minutes E Z N A SP Plant DNA Maxi Kit Protocols 24 Repeat Steps 21 23 for a second elution step Note This step may be performed using another 50 mL centrifuge tube to maintain a higher DNA concentration in the first eluate Alternatively DNA concentration can be increased by using the first eluate for the second elution step 25 Store DNA at 20 C 13 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 Problem Cause Solution Following precipitation with SP2 Buffer make Debris carryover ae sure no particulate material is transferred DNA pellet not completely dissolved before applying sample to column Ensure that DNA is dissolved in water before adding SP3 Buffer This may need repeated incubation at 65 C and vortexing Do not exceed suggested amount of starting Sample too viscous material Alternatively increase amounts of SP1 Buffer and SP2 Buffer Incomplete precipitation following addition of SP2 Buffer For both dried and fresh frozen samples obtain a fine homogeneous powder before adding SP1 Buffer Bgonlosiesttesaa Decrease amount of starting material or y increase amount of SP1 Buffer and SP2 Buffer DNA remains bound to Increase elution volume and incubate column col
12. umn at 65 C for 5 minutes before centrifugation Increase RCF or centrifugation time after addition of SP2 Buffer Incomplete disruption of starting material Low DNA yield Dilute SPW Wash Buffer by adding the DNA washed off appropriate volume of 100 ethanol prior to use Page 4 Follow the Optional Protocol for Column Equilibration prior to transferring the cleared lysate to the HiBind DNA Mini Column Add 3 mL 3M NaOH to the column prior to loading the sample Let sit for 4 minutes Centrifuge at 3 000 x g for 3 minutes Discard the filtrate SPW Wash Buffer must be at room Salt carryover Problems in temperature downstream Following the second wash step ensure applications Ethanol carryover that the column is dried by centrifugation at maximum speed for 10 minutes Column matrix lost binding capacity during storage 14 Ordering Information The following components are available for purchase separately Call Toll Free at 1 800 832 8896 SP1 Buffer 250 mL PDO86 SP2 Buffer 60 mL PD073 SP3 Buffer 100 mL PD074 SPW Wash Buffer 25 mL PDR045 Elution Buffer 100 mL PDR048 HiBind E Z N A and MicroElute are registered trademarks of Omega Bio tek Inc PCR is a patented process of Hoffman La Roche Use of the PCR process requires a license 15 Notes
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