Plant RNA/DNA Purification Kit - Protocol
Contents
1. Problem Possible Cause Solution and Explanation incomplete iysis ot Ensure that the homogenization step was done correctly POR y with the appropriate amount of Lysis Solution for the cells or tissue amount of cells or tissue Do not exceed the recommended amounts of starting materials The amount of starting material may need to Column has become clogged be decreased if the column shows clogging below the recommended levels See also Clogged Column below An alternative It is recommended that the Nucleic Acid Elution Buffer elution buffer was supplied with this kit be used for maximum RNA DNA Poor used recovery RNA DNA 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 added to the Ensure that 70 mL of 96 100 ethanol is added to the Nucleic Acid Wash supplied Nucleic Acid Wash Solution prior to use Solution Different tissues and cells have different RNA DNA Low RNA DNA contents and thus the expected yield of RNA DNA will content in cells or vary greatly from these different sources Please check tissues used literature to determine the expected RNA DNA content of your starting material 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 Procedim2. Lysis Solution please see the flow chart on page 4 The Lysis Solution contains detergents as well as large amounts of a chaotropic denaturant that will rapidly inactivate any RNases that are present A heat treatment is performed to ensure complete lysis Next Binding Solution is then added to the lysate followed by a short incubation on ice The lysate is then spun through the provided Filter Column in order to remove any debris Ethanol is then added to the clean lysate and the solution is loaded onto a spin column Norgen s resin binds nucleic acids in a manner that depends on ionic concentrations thus only the RNA and DNA will bind to the column while the proteins are removed in the flowthrough Next an optional step can be carried out in which the genomic DNA can be digested allowing for a more pure RNA sample to be isolated The bound nucleic acid is then washed three times with the provided Nucleic Acid Wash Solution in order to remove any impurities and the purified RNA and or DNA is eluted with the Nucleic Acid Elution Buffer The kit purifies all sizes of RNA from large mRNA and ribosomal RNA down to microRNA miRNA and small interfering RNA siRNA The purified RNA is of the highest integrity and can be used in a number of downstream applications including real time PCR reverse transcription PCR Northern blotting RNase protection and primer extension and expression array assays The genomic DNA is of the highest quality and c
3. It is recommended that gloves are changed frequently to avoid contamination e There should be designated solutions tips tubes lab coats pipettes 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 105 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 Flow Chart Procedure for Purifying Genomic DNA and Total RNA using Norgen s Plant RNA DNA Purification Kit Grind plant or fungi using liquid nitrogen Add Lysis Solution Incubate at 65 C Add Binding Solution Incubate on ice Transfer to Filter Column S SPIN Add Ethanol eo Bind to Spin Column al SPI Z Hol Wash three times with
4. and stored at room temperature These reagents should remain stable for at least 1 year in their unopened containers Precautions and Disclaimers This kit is designed for research purposes only 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 Plant RNA DNA Purification Kit Benchtop microcentrifuge 96 100 ethanol 70 ethanol Cell disruption tools such as mortar and pestle rotor stator homogenizer or bead mills Water bath or incubator heated to 65 C B mercaptoethanol Optional RNase free DNase Optional RNase A Optional Liquid nitrogen 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
5. and that it is incubated on ice for 5 minutes prior to the lysate spinning down the lysate Sampl tsoid Ensure that the sample is not too old as old samples p often yield only degraded DNA Genomic DNA is Sheared Samples should not be repeatedly frozen and thawed as Sample repeatedly frozen and thawed this tends to increase the likelihood of isolating degraded DNA 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 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 2014 Norgen Biotek Corp P124400 12 10
6. gt k 3430 Schmon Parkway Thorold ON Canada L2V 4Y6 i Phone 866 667 4362 e 905 227 8848 Fax 905 227 1061 BIOTEK si CORPORATION Email techsupport norgenbiotek com Plant RNA DNA Purification Kit Product Insert Product 24400 Norgen s Plant RNA DNA Purification Kit provides a rapid method for the isolation and purification of total RNA and DNA simultaneously from a single sample of plants The total RNA and DNA including genomic DNA and are both column purified in under 30 minutes using a single column It is often necessary to isolate total RNA and genomic DNA from a single plant sample such as for studies of gene expression mutant or transgenic plant characterization and host plant pathogen characterization Traditionally the RNA and DNA would be isolated from different aliquots of the same sample however this novel technology will allow for their simultaneous isolation from the same sample This will not only save time but will also be of a great benefit when isolating RNA and DNA from precious difficult to obtain or very small samples Furthermore gene expression analysis will be more reliable since the RNA and DNA are derived from the same sample therefore eliminating inconsistent results Norgen s Purification Technology Purification is based on spin column chromatography using Norgen s proprietary resin as the separation matrix The process involves first lysing the cells or tissue of interest with the provided
7. uL of Enzyme Incubation Buffer containing 15 uL of Norgen s RNase Free DNase Product 25710 to the column and centrifuge for 1 minute at 14 000 x g 14 000 RPM If using an alternative DNAse apply 100 uL of Enzyme Incubation Buffer containing 25 units of DNase to the column and centrifuge for 1 minute Note Ensure that the entire DNase solution passes through the column Repeat the step if needed At this point genomic DNA can be isolated instead of the total RNA If you wish to isolate RNA free genomic DNA apply 100 uL of Enzyme Incubation Buffer containing 10 units of RNase A user provided to the column and proceed as written below After the centrifugation in Step b pipette the flowthrough that is present in the collection tube back onto the top of the column Note Ensure Step 3c is performed in order to ensure maximum DNAse activity and to obtain maximum yields of RNA Incubate the whole unit at room temperature for 15 minutes Proceed to Step 4c 2 Column Wash without further centrifugation 4 Column Wash a Apply 400 uL of Nucleic Acid Wash Solution to the column and centrifuge for 1 minute 9 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 column with the collection tube Repeat steps 4a and 4b to wash column a second time
8. Wash Solution cl SPIN J Elute DNA and RNA with Elution Buffer SPIN 7 Purified Total RNA and Genomic DNA Notes Prior to Use 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 A variable speed centrifuge should be used for maximum kit performance If a variable speed centrifuge is not available a fixed speed centrifuge can be used however reduced yields may be observed Ensure that all solutions are at room temperature prior to use Prepare a working concentration of the Nucleic Acid Wash Solution by adding 70 mL of 96 100 ethanol provided by the user to the supplied bottle containing the concentrated Nucleic Acid Wash Solution This will give a final volume of 100 mL The label on the bottle has a box that may be checked to indicate that the ethanol has been added Optional For larger plant samples or samples with high starch or polysaccharide content we recommend the use of B mercaptoethanol during lysis Add 10 uL of B mercaptoethanol provided by the user to each 1 mL of Lysis Solution required B mercaptoethanol is toxic and should be dispensed in a fume hood Pre heat a water bath or an incubator to 65 C The optimal input of plant tissue is 50 mg or 5 x 10 plant cells However for most species up to 100 mg of tissue may be processed Both fresh and frozen plant samples can be used f
9. Wash column a third time by adding another 400 uL of Wash Solution and centrifuging for 1 minute Discard the flowthrough and reassemble the spin column with its collection tube Spin the column for 2 minutes in order to thoroughly dry the resin Discard the collection tube 5 Nucleic Acid Elution a Place the column into a fresh 1 7 mL Elution tube provided with the kit b Add 75 uL of Nucleic Acid Elution Buffer to the column Note If only RNA is being isolated reduce the volume of Nucleic Acid Elution Buffer to 100 uL c Centrifuge for 2 minutes at 200 x g 2 000 RPM followed by a 1 minute spin 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 nucleic acid recovery it is recommended that a second elution be performed into a separate microcentrifuge tube Repeat Steps 5b and 5c 6 Storage of DNA and RNA The purified nucleic acids may be stored at 20 C for a few days It is recommended that samples be placed at 70 C for long term storage Related Products Product Plant Fungi DNA Isolation kit 26200 Plant Fungi RNA Purification kit 25800 RNA DNA Protein Purification Kit 23500 RNA Protein Purification Kit 23000 RNase Free DNase Kit 25710 1kb RNA Ladder 15003 UltraRanger 1kb DNA Ladder 12100 Troubleshooting Guide
10. an be used in PCR reactions sequencing Southern blotting and SNP analysis Advantages e Fast and easy processing using rapid spin column format All columns for total RNA and genomic DNA purification provided Isolate total RNA from large rRNA down to microRNA miRNA No phenol or chloroform extractions Isolate high quality genomic DNA and total RNA Specifications Kit Specifications 50 ug for RNA 15 ug for genomic DNA Maximum Column Binding Capacity Maximum Column Loading Volume 650 uL Average Yields from 100 mg Peach Leaves 40 ug RNA 5 ug gDNA Raspberry Leaves 12 ug RNA 3 ug gDNA Strawberry Leaves 15 ug RNA 4 ug gDNA Grape Leaves 7 ug RNA 3 ug gDNA is All sizes including small RNA Size of RNA Purified lt 200 nt Maximum Amount of Starting Material Plant Tissues 100 mg Plant Cells 5x 10 Time to Complete 10 Purifications 30 minutes average yields will vary depending upon a number of factors including species growth conditions used and developmental stage Kit Components Component Product 24400 50 samples Lysis Solution 40 mL Binding Solution 7mL Nucleic Acid Wash Solution 30 mL Nucleic Acid Elution Buffer 9 mL Enzyme Incubation Buffer 6 mL Filter Columns 50 Spin Columns 50 Collection Tubes 100 Elution tubes 1 7 mL 50 Product Insert 1 Storage Conditions and Product Stability All solutions should be kept tightly sealed
11. bes Pipette the lysate into the Filter Column and spin for 2 minutes at 14 000 x g 14 000 RPM Transfer only the clear supernatant from the flow through into a DNAase free microcentrifuge tube not provided using a pipette Add an equal volume of 70 ethanol provided by the user to the lysate collected above 100 uL of ethanol is added to every 100 uL of lysate Vortex to mix Proceed to Step 2 2 Binding Nucleic Acids to Column Assemble a Spin Column grey O ring with one of the provided collection tubes Apply up to 600 uL of the clarified lysate with ethanol onto the column and centrifuge for 1 minute Discard the flowthrough and reassemble the spin column with the collection tube Note Ensure the entire lysate volume has passed through into the collection tube by inspecting the column If the entire lysate volume has not passed spin for an additional minute Depending on your lysate volume repeat step 2b if necessary 3 DNase Treatment Optional This optional step is carried out if genomic DNA free RNA is required It is recommended that Norgen s RNase Free DNase Kit Product 25710 be used for this step a Apply 400 uL of Nucleic Acid Wash Solution to the column and centrifuge for 2 minutes Discard the flowthrough 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 Apply 100
12. e not In order to maintain the integrity of the RNA it is performed quickly i important that the procedure be performed quickly enough impiroperstorageiof For short term storage RNA samples may be stored at propor g 20 C for a few days It is recommended that samples RNA is the purified RNA be stored at 70 C for longer term storage Degraded Frozen tissues or cell pellets were allowed to thaw prior to RNA isolation Tissue samples were frozen improperly Do not allow frozen tissues to thaw prior to cell disruption in order to ensure that the integrity of the RNA is not compromised Samples should be flash frozen in liquid nitrogen and transferred immediately to a 70 C freezer for long term storage Problem Possible Cause Solution and Explanation Insufficient Ensure that the appropriate amount of lysis buffer was solubilization of used for the amount of cells or tissue Ensure that the lysate was incubated at 65 C for 10 minutes Incubate cells or tissues l i E the lysis solution for an extra 5 minutes to assist in lysis ey nue The optimal input of plant tissue is 50 mg or 5 x 10 plant of cells or amount lis H f 100 fti of tissue exceeds ce an owever y most species up to 100 mg of tissue ios kit specifications ee aweans Too much cell debris in the lysate Ensure that most cell debris is removed in Step 1e supernatant Centrifuge Ensure that the centrifu
13. ge remains at room temperature g throughout the procedure Temperatures below 20 C temperature too ES f h h ou may cause precipitates to form that can cause the columns to clog RNA DNA was not Traces of salt from the binding step may remain in the washed three times sample if the column is not washed three times with RNA DNA with the provided Nucleic Acid Wash Solution Salt may interfere with does not Nucleic Acid Wash_ downstream applications and thus must be washed from perform well Solution the column in downstream Ensure that the dry spin under the Column Wash applications procedure is performed in order to remove traces of Emanolcaryover ethanol prior to elution Ethanol is known to interfere with many downstream applications Ensure that the appropriate amount of Lysis Solution was used for the amount of cells or tissue Ensure that the Incomplete lysis of lysate was incubated at 65 C for 10 minutes Incubate cells or tissue the lysis solution for an extra 5 minutes to assist in lysis Liquid nitrogen may be needed for lysis of challenging plant samples Yield of Ensure that centrifugation at 14 000 x g for 1 minute is RNA DNA is The DNA elution is Performed following the 2 minute centrifugation at 200 Low incoripl te xg Also ensure that the entire volume of Nucleic Acid z Elution Buffer passed through and is eluted from the column Binding Solution Ensure that the Binding Solution is added to the lysate was not added to
14. or this protocol Samples should be flash frozen in liquid nitrogen and transferred immediately to a 70 C freezer for long term storage Do not allow frozen tissues to thaw prior to grinding with the mortar and pestle in order to ensure that the integrity of the RNA is not compromised It is important to work quickly when purifying RNA This kit is provided with 2 separate columns When columns are removed from the labeled bags they are supplied in they can easily be identified as follows o Filter Columns contains a clear plastic O ring o Spin Columns contains a grey plastic O ring 1 Lysate Preparation a Place 100 mg of plant tissue or wet fungi into a mortar that contains liquid Nitrogen and grind into a powder Transfer the plant or fungi powder to a DNase free 1 7 mL microcentrifuge tube not provided and add 600 uL of Lysis Solution Alternatively other homogenization methods can be used with this procedure including a bead system If an alternative method is used add 600 uL of Lysis Solution to the sample immediately after homogenization and vortex for 20 seconds to mix Using a pipette transfer the lysate into an RNase free microcentrifuge tube provided by user Incubate the lysate at 65 C for 10 minutes Mix occasionally by inverting the tube a few times Add 100 uL of Binding Solution mix thoroughly and incubate for 5 minutes on ice Assemble a Filter Column clear O ring with one of the provided collection tu
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