E.Z.N.A.®HP Plant DNA Mini Kit - Omega Bio-Tek
Contents
1. 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 21 Notes 22 Notes 23 Notes 242. respectively Prepare the vacuum manifold according to manufacturer s instructions Connect the HiBind DNA Mini Column to the vacuum manifold Optional Protocol for Column Equilibration Add 100 uL 3M NaOH to the HiBind DNA Mini Column Let sit for 4 minutes at room temperature Turn on the vacuum to draw the NaOH through the column Turn off the vacuum RWN gt Transfer the cleared supernatant from Step 7 of the Dried or Fresh Frozen Protocols Pages 7 and 10 respectively or from Step 14 of the Lower DNA Content Protocol Page 15 by CAREFULLY aspirating it into the HiBind DNA Mini Column Be careful not to disturb the pellet and that no cellular debris is transferred to the HiBind DNA Mini Column Turn on the vacuum source to draw the sample through the column 17 10 11 12 13 18 E Z N A HP Plant DNA Mini Kit Protocols Turn off the vacuum 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 Turn on the vacuum source to draw the buffer through the column Turn off the vacuum Repeat Steps 7 9 for a second DNA Wash Buffer wash step Transfer the HiBind DNA Mini Column into a new 2 mL Collection Tube Centrifuge the empty column at maximum speed for 2 minutes Note It is important to dry the HiBind DNA Mini Column matrix before elution Residual ethanol may interfere with downstream applications T
3. buffers supplied with this kit are designed for the standard protocols Additional buffer amounts will be required for this protocol and can be purchased separately Please contact Omega Bio tek or its distributors for order information Materials and Equipment to be Supplied by User Microcentrifuge capable of at least 12 000 x g Swing bucket centrifuge capable of at least 3 000 x g Waterbath capable of 65 C e Vortexer Nuclease free 15 mL and 50 mL centrifuge tubes e Nuclease free 1 5 mL and 2 0 mL microcentrifuge tubes Chloroform e Isoamyl alcohol Isopropanol 100 ethanol Liquid nitrogen for freezing disrupting samples Sterile deionized water or 10 mM Tris pH 9 0 or 8 5 RNase A at 20 mg mL Optional 2 mercaptoethanol Before Starting Prepare the DNA Wash Buffer according to the instructions on Page 4 Prepare a mixture of chloroform isoamyl alcohol 24 1 Heat the sterile deionized water and Elution Buffer to 65 C Note Follow the suggestions for preparation of dried or fresh frozen specimens as outlined in the protocols above Pages 5 and 9 respectively Note the following limitations on sample size Dry Samples use a maximum of 200 mg ground tissue Fresh Frozen Samples use a maximum of 400 mg ground tissue 13 E Z N A HP Plant DNA Mini Kit Protocols Prepare 100 mg tissue in a 15 mL centrifuge tube not provided Add 9 mL CPL Buffer Vortex vigorously to mix Ma
4. form in CPL Buffer and CXD 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 D2485 02 100 mL per bottle 2 Prepare a mixture of chloroform isoamyl alcohol 24 1 Optional Prepare RNase A solution at 20 mg mL and aliquot into adequate portions Store each aliquot at 20 C and thaw before use Each sample will require 2 uL RNase A Protocol Selection Guide Protocol Ideal Sample For processing lt 50 mg powdered tissue Total DNA yield will vary depending on type and Dried Specimens quantity of sample Typically 10 50 ug DNA with aA Ay ratio of 1 7 1 9 can be isolated using 50 mg dried tissue For processing lt 200 mg fresh or frozen tissue Fresh or Frozen Specimens io Aaa Yield is similar to that for dried specimens For processing up to 200 mg dried or 450 mg fresh or frozen tissue Yields will vary according to sample size and whether dried or fresh Between 2 10 ug DNA can usually be obtained with this method Specimens with Lower DNA Content E Z N A HP Plant DNA Mini Kit Protocols E Z N A HP Plant DNA Mini Kit Protocol Dried Specimens This is the most robust method for isolation of total cellular mitochondrial chloroplast and genomic DNA Yields are usually sufficient for several tracks on a Southern blot for RFLP mapping Drying allows s
5. that may otherwise interfere with downstream applications Transfer the HiBind DNA Mini Column to a nuclease free 1 5 or 2 mL microcentrifuge tube not provided Add 100 uL Elution Buffer or sterile deionized water heated to 65 C Note Smaller elution volumes will increase DNA concentration but decrease yield Elution volumes greater than 200 uL are not recommended Centrifuge at 10 000 x g for 1 minute Repeat Steps 27 28 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 e Increase the elution volume e 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 E Z N A HP Plant DNA Mini Kit Protocols E Z N A HP Plant DNA Mini Kit Protocol Vacuum Method Note Please read through previous section of this book before using this protocol Materials and Equipment to be Supplied by User Vacuum Manifold Before Starting Prepare DNA Wash Buffer according to the Preparing Reagents section on Page 4 Heat Elution Buffer to 65 C Complete Steps 1 7 of either the Dried or Fresh Frozen Specimen Protocols or Steps 1 14 of the Protocol for Samples with Lower DNA Content Pages 5 9 and 13
6. to disturb the pellet or transfer any debris Add 150 uL CXD Buffer and 300 uL 100 ethanol Vortex to obtain a homogeneous mixture Note A precipitate may form upon addition of ethanol it will not interfere with DNA isolation Optional This the point to start the optional vacuum spin protocol See Page 17 for details 8 Insert a HiBind DNA Mini Column into a 2 mL Collection Tube Optional Protocol for Column Equilibration 10 Add 100 uL 3M NaOH to the HiBind DNA Mini Column Let sit for 4 minutes Centrifuge at maximum speed for 60 seconds Discard the filtrate and reuse the Collection Tube PUNA 10 11 12 13 14 15 16 17 18 19 E Z N A HP Plant DNA Mini Kit Protocols Transfer the entire sample including any precipitate that may have formed to the HiBind DNA Mini Column Centrifuge at 10 000 x g for 1 minute Discard the filtrate and the Collection Tube Transfer the HiBind DNA Mini Column to a new 2 mL Collection Tube Add 700 uL DNA Wash Buffer Note DNA Wash Buffer must be diluted with 100 ethanol prior to use Please refer to Page 4 or the bottle label for instructions Centrifuge at 10 000 x g for 1 minute Discard the filtrate and reuse the Collection Tube Repeat Steps 13 15 for a second DNA Wash Buffer wash step Centrifuge the empty HiBind DNA Mini Column at maximum speed for 2 minutes to dry the membrane Note It is critical to remove any tra
7. 4 15 16 17 18 19 E Z N A HP Plant DNA Mini Kit Protocols Transfer the entire sample including any precipitate that may have formed to the HiBind DNA Mini Column Centrifuge at 10 000 x g for 1 minute Discard the filtrate and the Collection Tube Transfer the HiBind DNA Mini Column to a new 2 mL Collection Tube Add 700 uL DNA Wash Buffer Note DNA Wash Buffer must be diluted with 100 ethanol prior to use Please refer to Page 4 or the bottle label for instructions Centrifuge at 10 000 x g for 1 minute Discard the filtrate and reuse the Collection Tube Repeat Steps 13 15 for a second DNA Wash Buffer wash step Centrifuge the empty HiBind DNA Mini Column at maximum speed for 2 minutes to dry the membrane Note It is critical to remove any trace of ethanol that may otherwise interfere with downstream applications Transfer the HiBind DNA Mini Column to a nuclease free 1 5 or 2 mL microcentrifuge tube not provided Add 100 uL Elution Buffer or sterile deionized water heated to 65 C Note Smaller elution volumes will increase DNA concentration but decrease yield Elution volumes greater than 200 uL are not recommended 20 21 22 E Z N A HP Plant DNA Mini Kit Protocols Centrifuge at 10 000 x g for 1 minute Repeat Steps 19 20 for a second elution step Note Any combination of the following steps can be used to help increase DNA yield After adding the Elution Buffe
8. E Z N A HP Plant DNA Mini Kit D2485 00 5 preps D2485 01 50 preps D2485 02 200 preps May 2013 E Z N A HP Plant DNA Mini Kit Table of Contents Introduction and OVervieW nn 2 Kit Contents Storage and Stability 3 Preparing Reagents see 4 Protocol for Dried Samples 5 Protocol for Fresh Frozen Samples 9 Protocol for Samples with Lower DNA Content 13 Vacuum Protocolos ass 17 Troubleshooting Guide 20 POSTING een 21 Manual Revision May 2013 02 OMEGA bio tek Innovations in nucleic acid isolation Introduction and Overview The E Z N A High Performance HP DNA Mini Kit is designed for efficient recovery of genomic DNA up to 60 kb in size from fresh frozen or dried plant tissue samples rich in polysaccharides or having a lower DNA content Up to 100 mg wet tissue or 50 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 inhibitors from plant tissue lysates Purified DNA is suitable for PCR restriction digestion and hybridization applications If using the E Z N A High Performance HP Plant DNA Mini Kit for the first time please read this booklet to become familiar with the procedures This procedure relies on the well established properties of the cationic detergent cetyl
9. ater before adding CXD Buffer and ethanol This may need repeated incubation at 650C and vortexing Do not exceed suggested amount of starting material Alternatively increase amounts of Buffers CPLand CXD and use two or more columns per sample Incomplete disruption of starting material Poor Iysis of sample DNA remains bound to column DNA washed off Salt carry over Ethanol carry over For both dry and fresh samples obtain a fine homogeneous powder before adding CPL Buffer Decrease amount of starting material or increase amount of CPL Buffer chlorosoamyl alcohol and CXD Buffer Increase elution volume to 200 uL and in cubate on column at 650C for 5 min before centrifugation Dilute DNA Wash Buffer by adding appropri ate volume of absolute ethanol prior to use page 3 Solution DNA Wash Buffer must be at room tempera ture Following the second wash spin ensure that the column is dried by centrifuging 2 min at maximum speed 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 Vacuum Manifold HiBind DNA Mini Columns 200 Elution Buffer 100 mL DNA Wash Buffer 100 mL RNase A 400 uL Part Number SSI 1210 00 SSI 1310 00 VAC 08 DNACOL 02 PDRO48 PSO10 AC117 HiBind
10. ce of ethanol that may otherwise interfere with downstream applications Transfer the HiBind DNA Mini Column to a nuclease free 1 5 or 2 mL microcentrifuge tube not provided Add 100 uL Elution Buffer or sterile deionized water heated to 65 C Note Smaller elution volumes will increase DNA concentration but decrease yield Elution volumes greater than 200 uL are not recommended 11 20 21 22 12 E Z N A HP Plant DNA Mini Kit Protocols Centrifuge at 10 000 x g for 1 minute Repeat Steps 19 20 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 e Increase the elution volume e 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 E Z N A HP Plant DNA Mini Kit Protocols E Z N A HP Plant DNA Mini Kit Protocol Specimens with Lower DNA Content This modified method allows rapid isolation of DNA from fresh frozen or dried specimens for sample types with lower DNA content or when larger yields are essential The procedure increases the amount of starting material so that DNA yields will generally be higher than those obtained with standard protocols above NOTE The
11. ke sure to disperse all clumps Note Process in sets of four to six tubes grind add CPL Buffer then proceed to Step 3 before starting another set Optional Add 10 ul 2 mercaptoethanol Vortex vigorously to mix 3 10 Its 12 14 Let sit at room temperature for 60 minutes Invert the samples twice during incuba tion Add 4 5 mL chloroform isoamyl alcohol 24 1 Vortex vigorously to mix Centrifuge at 3 000 x g for 10 minutes Carefully aspirate the top aqueous phase to a new 15 mL microcentrifuge tube making sure not to disturb the organic phase or transfer any debris Transfer the aqueous phase top to anew 15 mL centrifuge tube Add 0 7 volumes isopropanol Vortex to mix thoroughly Immediately centrifuge at 3 000 x g for 20 minutes Longer centrifugation does not improve yield Carefully aspirate and discard the supernatant making sure not to dislodge the DNA pellet Place inverted centrifuge tube on a paper towel for 1 minute to allow residual liquid to drain It is not necessary to dry the DNA pellet Add 400 uL sterile deionized water heated to 65 C Vortex to resuspend the pellet Note A brief incubation at 65 C may be necessary to effectively dissolve the DNA E Z N A HP Plant DNA Mini Kit Protocols 13 Add 20 uL RNase A 20 mg mL Vortex to mix thoroughly 14 Add 200 uL CXD Buffer and 400 uL 100 ethanol Vortex to obtain a homogeneous mixture Note A precipitate may form upon additio
12. n of ethanol it will not interfere with DNA isolation Optional This is the point to start the optional vacuum protocol See Page 17 for details 15 Insert a HiBind DNA Mini Column into a 2 mL Collection Tube Optional Protocol for Column Equilibration Add 100 uL 3M NaOH to the HiBind DNA Mini Column Let sit for 4 minutes at room temperature Centrifuge at maximum speed for 20 seconds Discard the filtrate and reuse the Collection Tube fe 0 E 16 Transfer 700 uL sample from Step 13 to the HiBind DNA Mini Column 17 Centrifuge at 10 000 x g for 1 minute 18 Discard the filtrate and reuse the collection tube 19 Repeat Steps 16 18 until all of the remaining sample including any precipitates that may have formed has been transferred to the HiBind DNA Mini Column 20 Transfer the HiBind DNA Mini Column to a new 2 mL Collection Tube 21 Add 700 uL DNA Wash Buffer Note DNA Wash Buffer must be diluted with 100 ethanol prior to use Please refer to Page 4 or the bottle label for instructions 15 22 23 24 25 26 27 28 29 30 16 E Z N A HP Plant DNA Mini Kit Protocols Centrifuge at 10 000 x g for 1 minute Discard the filtrate and reuse the Collection Tube Repeat Steps 21 23 for a second DNA Wash Buffer wash step Centrifuge the empty HiBind DNA Mini Column at maximum speed for 2 minutes to dry the membrane Note It is critical to remove any trace of ethanol
13. r incubate the column for 5 minutes e Increase the elution volume e 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 E Z N A HP Plant DNA Mini Kit Protocols E Z N A HP Plant DNA Mini Kit Protocol Fresh or Frozen Specimens This protocol is suitable for most fresh or frozen tissue samples However due to the tremendous variation in water and polysaccharide content of various fungi sample size should be limited to lt 200 mg The method isolates sufficient DNA for several tracks ona standard Southern assay To prepare samples collect tissue in a 1 5 or 2 mL microcentrifuge tube and freeze by dipping 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 one can allow liquid nitrogen to evaporate and then store 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
14. ransfer the HiBind DNA Mini Column to a nuclease free 1 5 or 2 mL microcentrifuge tube not provided 14 15 16 17 E Z N A HP Plant DNA Mini Kit Protocols Add 100 uL Elution Buffer or sterile deionized water heated to 65 C Note Smaller elution volumes will increase DNA concentration but decrease yield Elution volumes greater than 200 uL are not recommended Centrifuge at 10 000 x g for 1 minute Repeat Steps 14 15 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 19 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 Problem Problems in downstream applications 20 Carry over of debris DNA pellet not completely dissolved before applying sample to column Sample too viscous Following extraction with chloro isoamyl alcohol make sure no particulate material is transferred In Protocol C ensure that DNA is dissolved in w
15. repare 10 50 mg powdered dry tissue in a 1 5 or 2 mL microcentrifuge tube not provided 2 E Z N A HP Plant DNA Mini Kit Protocols Add 600 uL CPL Buffer Vortex vigorously to mix Make sure to disperse all clumps Note Process in sets of four to six tubes grind add CPL Buffer then proceed to Step 3 before starting another set Do not exceed 50 mg dried tissue Optional Add 10 ul 2 mercaptoethanol Vortex vigorously to mix Optional Add 2 uL RNase to the lysate before incubation to remove the RNA 3 Incubate at 65 C for 30 minutes Invert the samples twice during incubation Add 600 uL chloroform isoamyl alcohol 24 1 Vortex vigorously to mix Centrifuge at 210 000 x g for 10 minutes Transfer 300 uL aqueous phase top to a new microcentrifuge tube making sure not to disturb the pellet or transfer any debris Add 150 uL CXD Buffer and 300 uL 100 ethanol Vortex to obtain a homogeneous mixture Note A precipitate may form upon addition of ethanol it will not interfere with DNA isolation Optional This the point to start the optional vacuum spin protocol See Page 17 for details 8 Insert a HiBind DNA Mini Column into a 2 mL Collection Tube Optional Protocol for Column Equilibration Add 100 uL 3M NaOH to the HiBind DNA Mini Column Let sit for 4 minutes Centrifuge at maximum speed for 60 seconds Discard the filtrate and reuse the Collection Tube SNS 10 11 12 13 1
16. torage 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 50 mg of dried tissue into a microcentrifuge tube 2 mL tubes are recommended for processing of gt 50 mg tissue and grind using a pellet pestle Disposable Kontes pestles work well and are available from Omega Bio tek Cat 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 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 Materials and Equipment to be Supplied by User Microcentrifuge capable of at least 12 000 x g Waterbath capable of 65 C Vortexer Nuclease free 1 5 mL and 2 0 mL microcentrifuge tubes Chloroform Isoamyl alcohol Isopropanol 100 ethanol Optional RNase A at 20 mg mL e Optional 2 mercaptoethanol e Optional sterile deionized water Before Starting Prepare the DNA Wash Buffer according to the instructions on Page 4 Prepare a mixture of chloroform isoamyl alcohol 24 1 Heat the Elution Buffer or sterile deionized water to 65 C 1 P
17. trimethyl ammonium bromide CTAB in conjunction with the selective DNA binding of Omega Bio tek s HiBind matrix to isolate high quality DNA Samples are homogenized and Iysed in a high salt buffer containing CTAB and extracted with chloroform to remove polysaccharides and other components that interfere with many routine DNA isolations and downstream applications Binding conditions are adjusted and DNA is purified using a HiBind DNA Mini Columns 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 New in this Edition e This manual has been edited for content and redesigned to enhance user readability 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 D2485 00 D2485 01 D2485 02 Purifications En HiBind DNA Mini Columns 2 mL Collection Tubes CPL Buffer CXD Buffer DNA Wash Buffer Elution Buffer User Manual 10 mM Tris HCI pH 8 5 Storage and Stability All of the E Z N A HP Plant DNA Kit components are guaranteed for at least 12 months from the date of purchase when stored at room temperature During shipment or storage in cool ambient conditions precipitates may
18. wiping the surfaces clean between samples Materials and Equipment to be Supplied by User Microcentrifuge capable of at least 12 000 x g Waterbath capable of 65 C Vortexer e Nuclease free 1 5 mL and 2 0 mL microcentrifuge tubes Chloroform Isoamyl alcohol Isopropanol 100 ethanol Liquid nitrogen for freezing disrupting samples Optional RNase A at 20 mg mL e Optional 2 mercaptoethanol e Optional sterile deionized water Before Starting Prepare the DNA Wash Buffer according to the instructions on Page 4 Prepare a mixture of chloroform isoamyl alcohol 24 1 Heat the sterile deionized water and Elution Buffer to 65 C 1 Prepare 100 mgtissue in a 1 5 or 2 mL microcentrifuge tube not provided 2 E Z N A HP Plant DNA Mini Kit Protocols Add 500 uL CPL Buffer Vortex vigorously to mix Make sure to disperse all clumps Note Process in sets of four to six tubes grind add CPL Buffer then proceed to Step 3 before starting another set Do not exceed 50 mg dried tissue Optional Add 10 ul 2 mercaptoethanol Vortex vigorously to mix Optional Add 2 uL RNase to the lysate before incubation to remove the RNA 3 Incubate at 65 C for 15 minutes Invert the samples twice during incubation Add 800 uL chloroform isoamyl alcohol 24 1 Vortex vigorously to mix Centrifuge at 210 000 x g for 5 minutes Transfer 300 uL aqueous phase top to a new microcentrifuge tube making sure not
Download Pdf Manuals
Related Search