E.Z.N.A.®SP Plant DNA Midi Kit - Omega Bio-Tek
Contents
1. 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 1 Transfer up to 500 mg ground plant tissue to a 15 mL centrifuge tube not supplied 10 10 E Z N A SP Plant DNA Midi Kit Protocols Add 2 5 mL SP1 Buffer and 14 uL RNase A Vortex at maximum speed for 20 seconds to mix thoroughly Note Make sure to disperse all clumps by pipetting or vortexing Clumped tissue will not be lysed properly and will result in lower DNA yields Incubate at 65 C for 10 minutes Invert the tube several times during incubation Add 870 uL SP2 Buffer Vortex to mix thoroughly Let sit on ice for 10 minutes Centrifuge at 3 000 5 000 x g for 10 minutes at room temperature Carefully transfer the supernatant to a Homogenizer Midi Column Do not disturb the pellet Centrifuge at 3 000 5 000 x g for 5 minutes Note Longer centrifugation does not improve yield The Homogenizer Midi Column will remove most remaining precipitates and cell debris but a small amount may pass through and form a pellet in the collection tube Be careful not to disturb this pellet in Step 9 Carefully transfer the clea2. Buffer used in the Troubleshooting section is no longer included with this kit e Equilibration Buffer can be replaced with 3M NaOH provided by the user Kit Contents aa e oan Storage and Stability All of the E Z N A SP Plant DNA Midi Kit components are guaranteed for at least 12 months from the date of purchase when stored as follows Store RNase A 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 SP1 Buffer and SP3 Buffer Dissolve such deposits by warming the solution at 37 C and gently shaking Preparing Reagents Dilute SPW Wash Buffer with 100 ethanol as follows and store at room temperature Kit o 100 Ethanol to be Added Dilute SP3 Buffer with 100 ethanol as follows and store at room temperature 100 Ethanol to be Added E Z N A SP Plant DNA Midi Kit Protocols E Z N A SP Plant DNA Midi Kit Protocol for Dried Samples Materials and Equipment to be Supplied by User e Centrifuge capable of at least 3 000 x g e Nuclease free 15 mL and 50 mL high speed centrifuge tubes Incubator heat block or water bath capable of 65 C 100 ethanol Ice bucket or cryorack for centrifuge tubes e Optional Liquid nitrogen for freezing disrupting fresh samples Before Starting Prepare SPW Wash Buffer and SP3 Buffer according to the Preparing Reagents section on Page 4 Set an incubator
3. Ca OMEGA Innovations in nucleic acid isolation bio tek Product Manual E Z N A SP Plant DNA Midi Kit D5528 00 2 preps D5528 01 10 preps May 2013 For research use only Not intended for diagnostic testing E Z N A SP Plant DNA Midi Kit Table of Contents Introduction and OVEFVIEW cscssccsscsscsseccecssecnseessecneceneerses 2 Kit Contents Storage and Stability secsecssseceecseeeneens 3 Preparing Reagents ssseesssserssseeessseeesseeessseesssseossssosseeossesesssss 4 Recommended Settings or equivalent info cseee 5 Protocol for Dried SamMpleS ssssessssssseeserssssssssssseseesssssssseess 6 Protocol for Fresh Frozen SampleS ssssssssssssssseessssssssesse 6 Troubleshooting Guide sesserseeseesssesrsresrrsrrerrsrssrssrsssssess 14 Manual Revision May 2013 02 OMEGA bio tek Innovations in nucleic acid isolation Introduction and Overview The E Z N A SP Plant DNA Midi 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 500 mg wet tissue or 125 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 columns to eliminate polysaccharides phenolic compounds and enzyme inhibitors from plant tissue lysates
4. Purified DNA is suitable for PCR restriction digestion and hybridization applications There are no organic extractions thus reducing plastic waste and hands on time to allow multiple samples to be processed in parallel If using the E Z N A SP Plant DNA Midi 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 detergent Binding conditions are adjusted and the sample is transferred to a HiBind DNA Midi Column Three 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 Protocol Selection Choose the most appropriate protocol to follow Procedures are described for each of dried and fresh or frozen specimens Dry Specimens Ideal for processing 125 mg powdered tissue samples DNA yields Page 9 will vary and depend on genome size ploidy and sample age Fresh Frozen Specimens Page 13 Ideal for processing lt 500 mg fresh or frozen tissue Binding Capacity Each HiBind DNA Midi Column can bind up to 0 5 mg of genomic DNA Use More than 500 mg Fresh plant samples is not recommend New in this Edition e This manual has been edited for content and redesigned to enhance user readability Equilibration
5. and reuse the collection tube Repeat Steps 15 17 for a second SPW Wash Buffer wash step Centrifuge the empty HiBind DNA Midi Column at 5 000 x g for 10 minutes to dry the column matrix Note It is important to dry the HiBind DNA Midi Column matrix before elution Residual ethanol may interfere with downstream applications 11 20 21 22 23 24 25 12 E Z N A SP Plant DNA Midi Kit Protocols Transfer the HiBind DNA Midi Column to a new nuclease free 15 mL centrifuge tube not provided Add 400 500 uL Elution Buffer heated to 65 C directly to the center of the column matrix Note Smaller volumes will significantly increase DNA concentration but result in lower yields Let sit at room temperature for 5 minutes Note To increase DNA concentration add the buffer and incubate the column at 60 70 C for 5 minutes before elution Centrifuge at 3 000 5 000 x g for 3 minutes 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 a second elution 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 Following precipitation with SP2 Buffer ma
6. heat block or water bath to 65 C Heat Elution Buffer to 65 C Prepare an ice bucket or cryorack Drying allows storage of field specimens for prolonged period 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 125 mg dried tissue into a 15 mL centrifuge tube not supplied 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 cleaning 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 wiping the surface clean between samples A fine powder will ensure optimal DNA extraction and yield 1 Transfer 30 125 mg powdered dry tissue to a 15 mL centrifuge tube 2 Add 3 5 mL SP1 Buffer and 14 uL RNase A Vortex at maximum speed for 20 seconds to mix thoroughly Note Make sure to disperse all clumps by pipetting or vortexing Clumped tissue will not be lysed properly and will result in lower DNA yields 3 Incubate at 65 C for 30 60 minutes Invert the tube several times during incubation 10 E Z N A SP Plant DNA Midi Kit Protocols Add 1 25 mL SP2 Buffer Vortex to mix thoroughly Let sit on ice for 10 minutes Centrifuge at 3 000 5 000 x g for 10 minutes at room
7. ke Debris carryover ae sure no particulate material is transferred Do not exceed suggested amount of starting Sample too viscous material Alternatively increase the amount of SP1 Buffer and SP2 Buffer Incomplete precipitation following addition of SP2 Buffer For both dry and fresh samples obtain a fine homogeneous powder before adding SP1 Buffer Increase RCF or centrifugation time after the addition of SP2 Buffer Incomplete disruption of starting material Poor iysisottissue Decrease amount of starting material or y increase amount of SP1 Buffer and SP2 Buffer Low DNA yield DNA remains bound to Increase elution volume and incubate column column at 65 C for 5 minutes before centrifugation Dilute SPW Wash Buffer by adding DNA washed off appropriate volume of 100 ethanol prior to use Page 4 SPW Wash Buffer must be at room Salt carryover Problems in temperature downstream Following the second SPW Wash Buffer applications Ethanol carryover step ensure that the column is dried by centrifuging 10 minutes at maximum speed 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 PCR is a patented process of Hoffman La Roche Use of the PCR process requires a license 13 Notes
8. red lysate to a new 15 mL centrifuge tube not provided Do not disturb the pellet Measure the volume of the lysate for the next step Add 1 5 volumes SP3 Buffer Vortex immediately at maximum speed to obtain a homogenous mixture A precipitate may form at this point it will not interfere with DNA isolation Passing the mixture through a needle using a syringe or by pipetting up and down 10 15 times may break up the precipitates Note SP3 Buffer must be diluted with 100 ethanol before use Please see the instructions in the Preparing Reagents section on Page 4 E Z N A SP Plant DNA Midi Kit Protocols Optional Protocol for Column Equilibration 11 12 13 14 15 16 17 18 19 Add 1 mL 3M NaOH to a HiBind DNA Midi Column Let sit for 4 minutes Centrifuge at 3 000 x g for 3 minutes Discard the filtrate and reuse the Collection Tube FWN gt Transfer 3 5 mL cleared lysate including any precipitates from Step 10 to the HiBind DNA Midi Column Centrifuge at 3 000 5 000 x g for 5 minutes Discard the filtrate and reuse the collection tube Repeat Steps 11 13 until all of the cleared lysate has been transferred to the HiBind DNA Midi Column Add 3 mL SPW Wash Buffer Note SPW Wash Buffer must be diluted with 100 ethanol prior to use Please see the instructions in the Preparing Reagents section on Page 4 Centrifuge at 3 000 5 000 x g for 4 minutes Discard the filtrate
9. rifuge tube to maintain a higher DNA concentration in the first eluate Alternatively DNA concentration can be increased by using the first eluate for a second elution Store DNA at 20 C E Z N A SP Plant DNA Midi Kit Protocols E Z N A SP Plant DNA Midi Kit Protocol for Fresh Frozen Samples Materials and Equipment to be Supplied by User Centrifuge capable of at least 3 000 x g Nuclease free 15 mL and 50 mL high speed centrifuge tubes Water bath capable of 65 C 100 ethanol e Ice bucket or cryorack for centrifuge tubes Liquid nitrogen for freezing disrupting 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 Heat Elution Buffer to 65 C Prepare an ice bucket or cryorack Note Use extreme caution when handling liquid nitrogen This protocol is suitable for most fresh or frozen tissue samples allowing more efficient recovery of DNA However due to the tremendous variation in water and polysaccharide content of plants sample size should be limited to 500 mg Best results are obtained with young leaves or needles To prepare samples collect tissue in a 15 mL mortar and freeze by dipping in liquid nitrogen with a pair of tweezers to fill the tube Grind the tissue using a clean pestle Alternatively one can allow liquid nitrogen to evaporate and then store samples at 70 C
10. s Centrifuge at 3 000 5 000 x g for 5 minutes Discard the filtrate and reuse the collection tube Repeat Steps 11 13 until all of the cleared lysate has been transferred to the HiBind DNA Midi Column Add 3 mL SPW Wash Buffer Note SPW Wash Buffer must be diluted with 100 ethanol prior to use Please see the instructions in the Preparing Reagents section on Page 4 Centrifuge at 3 000 5 000 x g 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 HiBind DNA Midi Column at 5 000 x g for 10 minutes to dry the column matrix Note It is important to dry the HiBind DNA Midi Column matrix before elution Residual ethanol may interfere with downstream applications Transfer the HiBind DNA Midi Column to a new nuclease free 15 mL centrifuge tube not provided Add 400 500 uL Elution Buffer heated to 65 C directly to the center of the column matrix Note Smaller volumes will significantly increase DNA concentration but result in lower yields 22 23 24 25 E Z N A SP Plant DNA Midi Kit Protocols Let sit at room temperature for 5 minutes Note To increase DNA concentration add the buffer and incubate the column at 60 70 C for 5 minutes before elution Centrifuge at 3 000 5 000 x g for 3 minutes Repeat Steps 21 23 for a second elution step Note This step may be performed using another 50 mL cent
11. temperature Carefully transfer the supernatant to a Homogenizer Midi Column Do not disturb the pellet Centrifuge at 3 000 5 000 x g for 5 minutes Note Longer centrifugation does not improve yield The Homogenizer Midi Column will remove most remaining precipitates and cell debris but a small amount may pass through and form a pellet in the collection tube Be careful not to disturb this pellet in Step 9 Carefully transfer the cleared lysate to a new 15 mL centrifuge tube not provided Do not disturb the pellet Measure the volume of the lysate for the next step Add 1 5 volumes SP3 Buffer Vortex immediately at maximum speed to obtain a homogenous mixture A precipitate may form at this point it will not interfere with DNA isolation Passing the mixture through a needle using a syringe or by pipetting up and down 10 15 times may break up the precipitates Note SP3 Buffer must be diluted with 100 ethanol before use Please see the instructions in the Preparing Reagents section on Page 4 Optional Protocol for Column Equilibration 11 Add 1 mL 3M NaOH to a HiBind DNA Midi Column Let sit for 4 minutes Centrifuge at 3 000 x g for 3 minutes Discard the filtrate and reuse the Collection Tube T Transfer 3 5 mL cleared lysate including any precipitates from Step 10 to the HiBind DNA Midi Column 12 13 14 15 16 17 18 19 20 21 E Z N A SP Plant DNA Midi Kit Protocol
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