CleanAll DNA/RNA Clean-Up and Concentration Micro Kit
Contents
1. DNA does not Traces of salt from the binding step may remain in the sample if the column is not properly washed with the param wl E Wash Solution Ensure that the column is spun for 2 J wnetredit DNA minutes during the washing step Salt may interfere with care downstream applications and thus must be washed from applications se column The provided Elution Buffer has been optimized for ies eae endotoxin free recoveries If endotoxin free water is sed m for the elution ensure that the pH is between 7 and i The provided Elution Buffer has been optimized for Endotoxin endotoxin free recoveries The endotoxin free properties levels inthe A different Elution of the eluted DNA will be compromised if another elution eluted DNA Buffer was used buffer is used If a different Elution buffer other than the are slightly one provided is used the buffer should also be checked higher than for endotoxin levels 0 1 EU ug i DNA If the initial input DNA had extremely high endotoxin The endotoxin levels of the input were extremely high levels the levels may not be completely reduced to 0 1 EU ug of DNA or less In this case the eluted DNA could be applied to a second column and the procedure repeated in order to further reduce the endotoxin levels 12 Problem Possible Cause Solution and Explanation Do not exceed the recommended amounts of starting Column has materials T2. Improper storage of the purified RNA For short term storage RNA samples may be stored at 20 C for a few days It is recommended that samples be stored at 70 C for longer term storage 13 Problem Possible Cause Solution and Explanation RNA does not RNA was not washed twice with the provided Wash Traces of salt from the binding step may remain in the sample if the column is not washed twice times with Wash Solution Salt may interfere with downstream perform well Solution applications and thus must be washed from the column in downstream Ensure that the dry spin under the Column Wash applications Ethanol c rtvov r procedure is performed in order to remove traces of y ethanol prior to elution Ethanol is known to interfere with many downstream applications DNA or l Genome Large amounts of Perform RNAse free DNasel digestion on the RNA DNA starting material sample before clean up according to the protocol contamination used provided in Appendix A Related Products Product Total RNA Purification Kit 17200 Cytoplasmic amp Nuclear RNA Purification Kit 21000 Leukocyte RNA Purification Kit 21200 microRNA Purification Kit 21300 100b RNA Ladder 15002 1kb RNA Ladder 15003 Technical Support Contact our Technical Support Team between the hours of 8 30 and 5 30 Eastern Standard Time at 905 227 8848 or Toll Free at 1 866 667
3. 4362 Technical support can also be obtained from our website www norgenbiotek com or through email at techsupport norgenbiotek com 3430 Schmon Parkway Thorold ON Canada L2V 4Y6 Phone 905 227 8848 Fax 905 227 1061 Toll Free in North America 1 866 667 4362 2008 Norgen Biotek Corp P123800 8 14
4. DNA Staining Including CsCl gradients Intercalating dyes such as ethidium bromide and SYBR Green salts Applications Requiring RNA Cleanup Contaminants Removed DNase treatment DNase salts In vitro transcription DNA template nucleotides enzymes salts RNA Probe Labeling Radioactive Fluorescent or Digoxigenin RNA polymerase nucleotides salts label or coupling reagent Organic based RNA Isolation such as Trizol based lysate Phenol chloroform Advantages e Versatile performance for cleaning up all nucleic acids Fast and easy processing using rapid spin column format Suitable for all sizes of RNA from large rRNA down to microRNA miRNA Suitable for all sizes of DNA from large gDNA down to small PCR fragments Optional endotoxin removal No phenol or chloroform extractions Cleanup nucleic acid from a variety of isolation methods and enzymatic reactions Kit Components Component Product 23800 50 samples Binding Solution 60 mL Wash Solution 27 mL Elution Buffer 15 mL Micro Spin Columns 50 Collection Tubes 50 Elution tubes 1 7 mL 50 Product Insert 1 Storage Conditions and Product Stability All solutions should be kept tightly sealed and stored at room temperature These reagents should remain stable for at least 1 year in their unopened containers Precautions and Disclaimer This kit is designed for research purposes onl
5. Solution This will give a final volume of 81 mL The label on the bottle has a box that may be checked to indicate that the ethanol has been added Prepare an appropriate amount of Binding Solution by adding 10 uL of B mercaptoethanol provided by the user to each 1 mL of Binding Solution required B mercaptoethanol is toxic and should be dispensed in a fume hood It is recommended that no more than 50 ug of RNA to be used per cleanup The maximum volume of RNA sample that can be processed is 200 uL It is important to work quickly during this procedure This kit purifies RNA with minimal amounts of DNA contamination However an optional protocol is provided in Appendix A for maximum removal of residual DNA that may affect sensitive downstream applications such quantitative PCR 1 Sample Preparation a Adjust the volume of the RNA sample to 100 uL by adding RNase free or DEPC treated water It is recommended that no more than 50 ug of RNA be used for each column Note If an input volume between 100 and 200 uL is used adjust the sample volume to 200 uL maximum allowable with RNase free or DEPC treated water In this case use the volumes indicated in bold in the bracket in Steps 1b and 1c Add 250 uL or 500 uL of Binding Solution to the RNA sample Mix by vortexing Add 200 uL or 400 uL of 95 100 ethanol provided by the user to the mixture from step 1b Mix by vortexing for 10 seconds 2 Binding to Column a Assemble
6. for 2 minutes at 14 000 x g Discard the flowthrough and reassemble the unit Spin the column for an additional 1 minute at 14 000 x g in order to completely dry the resin Discard the collection tube 4 Elution of Clean DNA a b Assemble the column with one of the provided 1 7 mL Elution tubes Elute DNA with the provided Elution Buffer according the following table 10 000 bp or larger 1000 bp to 10 000 bp Smaller than 1000 bp Recommended Elution Volume ae eo ur Op Minimum Elution Volume 50 uL 20 uL 20 uL 2 minutes at 200 x g 2 minutes at 200 x g Elution Speed followed by followed by 1 minute at 14 000 x g 1 minute at 14 000 xg 1 minute at 14 000 x g c Optional An additional elution can be performed by repeating steps 4a and 4b This elution should be collected into a separate tube to avoid diluting the DNA solution in the first elution C Protocol for RNA Clean up and Concentration from Enzymatic Reactions or Other Non Phenol Guanidine Based RNA Isolation Methods All centrifugation steps are carried out in a benchtop microcentrifuge at 14 000 x g 14 000 RPM except where noted All centrifugation steps are performed at room temperature Notes Prior to Use Ensure that all solutions are at room temperature prior to use Prepare a working concentration of the Wash Solution by adding 54 mL of 95 ethanol provided by the user to the supplied bottle containing the concentrated Wash
7. free microcentrifuge tube not provided Note the volume b Add one volume of 70 ethanol provided by the user to the fraction from step 1a Mix by vortexing for 10 seconds 2 Binding to Column a Assemble a column with one of the provided collection tubes b Apply up to 600 uL of the RNA mixed with the ethanol from Step 1b onto the column and centrifuge for 1 minute c Discard the flowthrough Reassemble the spin column with its collection tube d If the volume of RNA mix is greater than 600 uL repeat Steps 2b and 2c until all the remaining RNA mix has passed through the column 3 Column Wash a Apply 500 uL of Wash Solution to the column and centrifuge for 1 minute Note Ensure the entire wash solution has passed through into the collection tube by inspecting the column If the entire wash volume has not passed spin for an additional minute Discard the flowthrough and reassemble the spin column with its collection tube Repeat steps 3a and 3b to wash the column a second time e Wash column a third time by adding another 500 uL of Wash Solution and centrifuging for 2 minutes f Ensure that the column is dry Spin for an additional minute if necessary g Discard the collection tube with the flowthrough a9 4 RNA Elution a Place the column into a fresh 1 7 mL Elution tube provided with the kit b Add 50 uL of Elution Buffer to the column 10 Note For higher concentrations of RNA a lower elution volume may b
8. up to 600 uL of the DNA sample from Step 1b onto the column and centrifuge for 1 minute Discard the flowthrough Reassemble the spin column with its collection tube If the volume of the DNA sample is greater than 600 uL repeat Step 2b and 2c until all the remaining DNA sample has passed through the column 3 Column Wash a Apply 500 uL of Wash Solution to the column assembly and centrifuge the unit for 2 minutes at 14 000 x g Discard the flowthrough and reassemble the unit Spin the column for an additional 1 minute at 14 000 x g in order to completely dry the resin Discard the collection tube Ensure that the column is dry Spin for an additional minute if necessary Discard the collection tube with the flowthrough 4 Elution of Clean DNA a Assemble the column with one of the provided 1 7 mL Elution tubes b Elute DNA with the provided Elution Buffer according the following table 10 000 bp or larger 1000 bp to 10 000 bp Smaller than 1000 bp Recommended Elution Volume TOSHE popl age Minimum Elution Volume 50 uL 20 uL 20 uL 2 minutes at 200 x g 2 minutes at 200 x g Elution Speed followed by 1 minute followed by 1 minute 1 minute at 14 000 x g at 14 000 xg at 14 000 xg c Optional An additional elution can be performed by repeating steps 3a and 3b This elution should be collected into a separate tube to avoid diluting the DNA solution in the first elution B Protocol for DNA C
9. a column with one of the provided collection tubes b Apply up to 600 uL of the RNA sample with the ethanol from Step 1c onto the column and centrifuge for 1 minute c Discard the flowthrough Reassemble the spin column with its collection tube d If the volume of the RNA sample is greater than 600 uL repeat Step 2b and 2c until all the remaining RNA sample has passed through the column 3 Column Wash a Apply 500 uL of Wash Solution to the column and centrifuge for 1 minute Note Ensure the entire wash solution has passed through into the collection tube by inspecting the column If the entire wash volume has not passed spin for an additional minute b Discard the flowthrough and reassemble the spin column with its collection tube c Wash column a second time by adding another 500 uL of Wash Solution and centrifuging for 2 minutes d Ensure that the column is dry Spin for an additional minute if necessary e Discard the collection tube with the flowthrough 4 RNA Elution a Place the column into a fresh 1 7 mL Elution tube provided with the kit b Add 50 uL of Elution Buffer to the column Note For higher concentrations of RNA a lower elution volume may be used A minimum volume of 20 uL is recommended c Centrifuge for 2 minutes at 200 x g 2 000 RPM followed by 1 minute at 14 000 x g 14 000 RPM Note the volume eluted from the column If the entire volume has not been eluted spin the column at 14 000
10. anol Bind to column SPIN Wash two times Bind to column with Wash Solution SPIN SPIN Elute DNA with Wash two times Elution Buffer with Wash Solution SPIN SPIN ae EO A aO aM Elute RNA with Purified DNA Elution Buffer SPIN A O A OA wo A aa Purified RNA A Protocol for DNA Cleanup Notes Prior to Use Ensure that all solutions are at room temperature prior to use Prepare a working concentration of the Wash Solution by adding 54 mL of 95 ethanol provided by the user to the supplied bottle containing the concentrated Wash Solution This will give a final volume of 81 mL The label on the bottle has a box that may be checked to indicate that the ethanol has been added It is recommended that no more than 10 ug of DNA to be used per cleanup The maximum volume of DNA sample that can be processed is 200 uL 1 Sample Preparation a b Adjust the volume of the DNA sample to 100 uL by adding molecular biology grade water It is recommended that no more than 10 ug of DNA be used for each column Note If an input volume between 100 and 200 uL is used adjust the sample volume to 200 uL maximum allowable with molecular biology grade water In this case use the volumes indicated in bold in the bracket in Steps 1b Add 500 uL or 1 mL of Binding Solution to the DNA sample Mix by vortexing 2 Binding to Column a b c d Assemble a column with one of the provided collection tubes Apply
11. cok 3430 Schmon Parkway K Thorold ON Canada L2V 4Y6 z Phone 866 667 4362 905 227 8848 Fax 905 227 1061 BIOTEK ad CORPORATION Email techsupport norgenbiotek com CleanAll DNA RNA Clean up and Concentration Micro Kit Product 23800 Product Insert Norgen s CleanAll DNA RNA Clean up and Concentration Micro Kit provides a rapid method for the purification cleanup and concentration of DNA or RNA from different isolation methods or upstream applications This kit purifies all sizes of DNA from small PCR products native or linearized plasmids to genomic DNA The kit can be used as an alternative to organic extraction and ethanol precipitation to clean up various enzymatic reactions It effectively removes PCR by products including primers dimers enzymes and unincorporated nucleotides The purified DNA is fully compatible with restriction enzyme digestion ligation labeling PCR and sequencing This kit also purifies RNA from phenol guanidine based protocols or from various upstream enzymatic reactions such as DNase treatment labeling and in vitro transcription The kit purifies all sizes of RNA from large mRNA and ribosomal RNA down to microRNA miRNA and small interfering RNA siRNA The RNA is preferentially purified from other reaction components such as proteins and nucleotides without the use of phenol or chloroform The purified RNA is of the highest integrity and can be used in a number of downstream applications inclu
12. d in the flowthrough The bound nucleic acid is then washed two times with the provided Wash Solution in order to remove any remaining impurities The purified DNA or RNA is eluted with Elution Buffer The purified DNA or RNA is of the highest integrity and can be used in a number of downstream applications Specifications Kit Specifications Column Binding Capacity 50 ug for RNA 10 ug for DNA Maximum Column Loading Volume 600 uL Size of RNA Purified All sizes including small RNA lt 200 nt Size of DNA Purified gt 100 bp Maximum Amount of Starting Material 50 ug of RNA 10 ug of DNA Time to Complete 10 Purifications 20 minutes Minimum Elution Volume 20 uL Average Recovery gt 90 for RNA gt 90 for DNA from 100 bp to 10 kbp gt 75 for DNA gt 10 kbp Kit Applications Applications Requiring DNA Cleanup Contaminants Removed Restriction Enzyme Digestion Restriction enzymes salts DNA Modifications Klenow T4 Polynucleotide Kinase Alkaline Phosphatase DNA modification enzymes nucleotides salts PCR Reactions DNA polymerases nucleotides salts primers mineral oils DNA Probe Labeling Radioactive Fluorescent or Digoxigenin DNA modification enzymes or polymerase nucleotides label or coupling reagent salts primers Ligation Ligase nucleotides salts Transfection Microinjection Endotoxin
13. ding end point or quantitative reverse transcription PCR Northern blotting RNase protection and primer extension and expression array assays Norgen s CleanAll DNA RNA Clean up and Concentration Micro Kit also provides a protocol for the rapid removal of endotoxins from previously purified DNA down to 0 1 EU ug DNA or less Endotoxins also known as lipopolysaccharides are cell membrane components of Gram negative bacteria such as E coli Endotoxins are released during the lysis step of plasmid purification and significantly reduce transfection efficiencies in endotoxin sensitive cell lines Therefore the removal of endotoxins from plasmid preparations is often necessary prior to the use of the DNA in downstream applications Norgen s Purification Technology Purification is based on spin column chromatography using Norgen s proprietary resin as the separation matrix The DNA or RNA is preferentially purified from other cellular components such as proteins without the use of phenol or chloroform The process involves first mixing the nucleic acid samples or enzymatic reactions containing DNA or RNA with Binding Solution please see the flow chart on page 5 For RNA purification ethanol is then added and the mixture is loaded onto a spin column Norgen s resin binds nucleic acids in a manner that depends on ionic concentrations Thus only the nucleic acids will bind to the column while the contaminating proteins or nucleotides will be remove
14. e RNA sample 4 Incubate at 25 to 30 C for 15 minutes 5 Proceed directly to Protocol A Protocol for RNA Clean up and Concentration from Enzymatic Reactions or Other Sample Preparation Methods 11 Troubleshooting Guide Problem Possible Cause Solution and Explanation ae Binding of the DNA is dependent on both pH and salt ras is concentration Ensure that an appropriate amount of S gee Binding Solution was used for the volume of the DNA inefficient Input The appropriate The Wash Solution has been specifically designed to amount of ethanol f was not add d ic contain the appropriate amount of components Ensure the Wash that the Wash Solution was prepared using the correct Concentrate amount of ethanol Poor DNA Binding Solunar Traces of salt left on the column from the binding step recovery was not completely removed in the wash step may interfere with the elution of the DNA Ensure that the column is washed with the Wash Solution Proper Elution Buffer was not used The provided Elution Buffer has been optimized for high elution recoveries If water or TE buffer is used instead ensure the pH is around 8 Elution Buffer was not placed directly onto the resin It is important that the Elution Buffer be placed directly onto the resin as this helps to increase recovery by ensuring an even passing of the buffer through the resin Do not pipette the Elution Buffer onto the side of the column
15. e used A minimum volume of 20 uL is recommended c Centrifuge for 2 minutes at 200 x g 2 000 RPM followed by 1 minute at 14 000 x g 14 000 RPM Note the volume eluted from the column If the entire volume has not been eluted spin the column at 14 000 x g 14 000 RPM for 1 additional minute Note For maximum RNA recovery it is recommended that a second elution be performed into a separate microcentrifuge tube Repeat Steps 4b and 4c 5 Storage of RNA The purified RNA sample may be stored at 20 C for a few days It is recommended that samples be placed at 70 C for long term storage Appendix A Optional DNA Removal in Solution Followed by RNA Clean up and Concentration Norgen s CleanAll DNA RNA Clean up and Concentration Micro Kit purifies RNA with minimal amounts of DNA contamination An optional protocol is provided below for maximum removal of residual DNA that may affect sensitive downstream applications such quantitative PCR It is recommended that an RNase free DNase be used 1 Adjust the volume of the RNA sample to be treated to 80 uL with RNase free water 2 Prepare a working stock of 0 5 Kunitz unit uL RNase free DNase solution according to the manufacturer s instructions Alternatively dissolve or dilute stock DNase lin a reaction buffer 40 mM Tris pH 7 0 10 mM MgCl and 3 mM CaCl made RNase free to give a final concentration of 0 5 Kunitz unit uL 3 Add 20 uL of 0 5 Kunitz unit uL DNase to th
16. he amount of starting material may need to become clogged be decreased if the column shows clogging below the 99 recommended levels See also Clogged Column below a ya It is recommended that the Elution Buffer supplied with ot eed this kit be used for maximum RNA recovery Ethanol was not Ensure that the appropriate amount of ethanol is added added to the lysate to the lysate before binding to the column Ethanol was not E hat 40 mL of 95 ethanol is added to th added to the Wash nsure t at 40 m of 95 et anol is added to the Solution supplied Wash Solution prior to use High amounts of Ensure that no more than 50 ug of RNA are used as RNA as input input High amounts of The lysate may be passed through a 25 gauge needle Clogged genomic DNA attached to a syringe 5 10 times in order to shear the Column present in sample genomic DNA prior to loading onto the column Centrifuge Ensure that the centrifuge remains at room temperature tem a o0 throughout the procedure Temperatures below 15 C iow P may cause precipitates to form that can cause the columns to clog RNases may be introduced during the use of the kit RNase Ensure proper procedures are followed when working contamination with RNA Please refer to Working with RNA at the beginning of this user guide Procedure not beg se RNA is performed quickly In order to maintain the integrity of the RNA it is Degraded enough important that the procedure be performed quickly
17. leanup with Endotoxin Removal Notes Prior to Use Ensure that all solutions are at room temperature prior to use Prepare a working concentration of the Wash Solution by adding 54 mL of 95 ethanol provided by the user to the supplied bottle containing the concentrated Wash Solution This will give a final volume of 81 mL The label on the bottle has a box that may be checked to indicate that the ethanol has been added It is recommended that no more than 10 ug of DNA to be used per cleanup The maximum volume of DNA sample that can be processed is 200 uL 1 Sample Preparation a b c Transfer up to 100uL of DNA into a microcentrifuge tube Add 500 uL of Binding Solution to the DNA and mix well by inversion or vortexing Assemble a spin column with a provided collection tube Add the DNA solution to the top of the column Let the column assembly stand for 5 minutes at room temperature Note If dripping occurs into the collection tube during the 5 minute incubation period just proceed with protocol as written After 5 minutes add 60 uL of isopropanol to the liquid on the column Close lid and vortex column assembly gently to mix 2 Binding to Column a b Spin the column at 14 000 x g for 1 minute in a microcentrifuge Discard the flowthrough and reassemble the spin column with its collection tube 3 Washing Bound DNA a b c Apply 500 uL of Wash Solution to the column assembly and centrifuge the unit
18. tes etc for RNA only e All RNA solutions should be prepared using at least 0 05 DEPC treated autoclaved water or molecular biology grade nuclease free water e Clean all surfaces with commercially available RNase decontamination solutions e When working with purified RNA samples ensure that they remain on ice during downstream applications Procedures All centrifugation steps are carried out in a benchtop microcentrifuge Various speeds are required for different steps so please check your microcentrifuge specifications to ensure that it is capable of the proper speeds All centrifugation steps are performed at room temperature The correct rom can be calculated using the formula RPM ___RCF_ _ 1 118 x 10 r where RCF required gravitational acceleration relative centrifugal force in units of g r radius of the rotor in cm and RPM the number of revolutions per minute required to achieve the necessary g force Since Norgen s CleanAll DNA RNA Clean up and Concentration Micro Kit uses common solutions for DNA and RNA clean up it is important for user to maintain an RNase free environment if both procedures are to be carried out either simultaneously or separately Flow Chart Procedure for Purifying DNA RNA using Norgen s CleanAll DNA RNA Clean up and Concentration Micro Kit Obtain Nucleic Acid Sample RNA or DNA and add Binding Solution Vortex to mix ZN For RNA Purification For DNA Purification Add eth
19. x g 14 000 RPM for 1 additional minute Note For maximum RNA recovery it is recommended that a second elution be performed into a separate microcentrifuge tube Repeat Steps 4b and 4c 5 Storage of RNA The purified RNA sample may be stored at 20 C for a few days It is recommended that samples be placed at 70 C for long term storage D Protocol for RNA Clean up and Concetration from Phenol Guanidine based RNA Trizol or Tri Reagent Isolation Methods All centrifugation steps are carried out in a benchtop microcentrifuge at 14 000 x g 14 000 RPM except where noted All centrifugation steps are performed at room temperature Notes Prior to Use e Ensure that all solutions are at room temperature prior to use e Prepare a working concentration of the Wash Solution by adding 54 mL of 95 ethanol provided by the user to the supplied bottle containing the concentrated Wash Solution This will give a final volume of 81 mL The label on the bottle has a box that may be checked to indicate that the ethanol has been added e It is recommended that no more than 50 ug of RNA to be used per cleanup e It is important to work quickly during this procedure 1 Sample Preparation a Isolate RNA using a phenol guanidine based reagent such as Trizol or Tri Reagent according to manufacturer s instruction After the separation of the aqueous and organic phases collect the upper aqueous fractions containing the RNA into a new RNase
20. y It is not intended for human or diagnostic use Ensure that a suitable lab coat disposable gloves and protective goggles are worn when working with chemicals For more information please consult the appropriate Material Safety Data Sheets MSDSs These are available as convenient PDF files online at www norgenbiotek com Customer Supplied Reagents and Equipment You must have the following in order to use the CleanAll DNA RNA Clean up and Concentration Micro Kit For All Protocols e Benchtop microcentrifuge e Microcentrifuge tubes e 96 100 ethanol For Endotoxin Removal e lsopropanol For RNA Clean up and Concentration e mercaptoethanol For RNA Clean up and Concentration from Phenol Guanidine based RNA Trizol or Tri Reagent Isolation Methods e 70 ethanol Working with RNA RNases are very stable and robust enzymes that degrade RNA Autoclaving solutions and glassware is not always sufficient to actively remove these enzymes The first step when preparing to work with RNA is to create an RNase free environment The following precautions are recommended as your best defense against these enzymes e The RNA area should be located away from microbiological work stations e Clean disposable gloves should be worn at all times when handling reagents samples pipettes disposable tubes etc It is recommended that gloves are changed frequently to avoid contamination e There should be designated solutions tips tubes lab coats pipet
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