43400 - Protocol (50 prep)
Contents
1.2. 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 Notes Prior to Use e Refer to manufacturer s product documentation for specific instructions on venipuncture technique blood collection storage and shipment conditions and safety information e 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 e 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 e Ensure that all solutions are at room temperature prior to use e Prepare a working concentration of the Wash Solution by adding 50 mL of 95 ethanol provided by the user to the supplied bottle containing the concentrated Wash Solution This will give a final volume of 72 mL The label on the bottle has a box that may be checked to indicate that the ethanol has been added e It is important to work quickly during this procedure e Blood of all human and animal subjects is considered potentially infectious All necessary precautions recommended by the appropriate authorities in the country of use should be taken when working with whole blood 1 Lysate Prepa
3. mL Mini Spin Columns 50 Collection Tubes 50 Elution tubes 1 7 mL 50 Product Insert 1 Precautions and Disclaimers This kit is designed for research purposes only It is not intended for 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 Blood of all human and animal subjects is considered potentially infectious All necessary precautions recommended by the appropriate authorities in the country of use should be taken when working with whole blood The Lysis Solution contains guanidinium salts and should be handled with care Guanidinium salts form highly reactive compounds when combined with bleach thus care must be taken to properly dispose of any of these solutions Customer Supplied Reagents and Equipment You must have the following in order to use the Total RNA Purification Kit e Benchtop microcentrifuge e Swing bucket centrifuge e 50 mL conical tubes e 95 100 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
4. 3430 Schmon Parkway gt N Thorold ON Canada L2V 4Y6 Phone 866 667 4362 e 905 227 8848 Fax 905 227 1061 BIOTEK amp CORPORATION Email techsupport norgenbiotek com Preserved Blood RNA Purification Kit Product Insert for use with Tempus Blood RNA Tubes Product 43400 Norgen s Preserved Blood RNA Purification Kit provides a rapid method for the isolation and purification of total RNA from blood that has been preserved using Tempus Blood RNA Tubes 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 cellular components such as proteins 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 including real time PCR reverse transcription PCR Northern blotting RNase protection and primer extension and expression array assays Norgen s Purification Technology Purification is based on spin column chromatography using Norgen s proprietary resin as the separation matrix The RNA is preferentially purified from other cellular components such as proteins without the use of phenol or chloroform The process involves first lysing the preserved blood using the provided Lysis Solution please see the flow chart on page 4 Ethanol is then added to the lysate and the solution is loaded onto a spin column Norg
5. 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 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 Flowchart Procedure for Purifying Total RNA from Preserved Blood using Norgen s Preserved Blood RNA Purification Kit Pellet cells from preserved blood sample Add Lysis Solution 4 Add Ethanol Bind to column SPIN D lt lt Wash three times with Wash Solution SPIN Elute RNA with Elution Solution SPIN a Purified Total RNA 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
6. en s Preserved Blood RNA Purification Kit isolates total RNA with minimal amounts of genomic DNA contamination However an optional On Column DNA Removal Protocol is provided in Appendix A for maximum removal of residual DNA that may affect sensitive downstream applications It is recommended that Norgen s RNase Free DNase Kit Product 25710 be used for this step This step should be performed at this point in the protocol 3 Column Wash a Apply 400 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 Repeat steps 3a and 3b to wash column a second time d Wash column a third time by adding another 400 uL of Wash Solution and centrifuging for 1 minute e 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 9 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 Solution to the column 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 50 uL has not been eluted spi
7. en s resin binds RNA in a manner that depends on ionic concentrations Thus only the RNA will bind to the column while the contaminating proteins will be removed in the flowthrough or retained on the top of the resin The bound RNA is then washed with the provided Wash Solution in order to remove any remaining impurities and the purified total RNA is eluted with the Elution Solution The purified RNA is of the highest integrity and can be used in a number of downstream applications Specifications Kit Specifications Maximum Column Binding Capacity 50 ug Maximum Column Loading Volume 650 uL Size of RNA Purified All sizes including small RNA lt 200 nt Time to Complete 10 Purifications 20 minutes Average Yield 5 25 ug per 3 mL preserved human blood Advantages e Fast and easy processing of preserved blood using rapid spin column format e Isolate total RNA from large rRNA down to microRNA miRNA e No phenol or chloroform extractions e Isolate high quality total RNA 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 Tempus is a registered trademark of Life Technologies Kit Components Component Product 43400 50 preps Tapaa Blood RNA Tube EE Lysis Solution 40 mL Wash Solution 22 mL Elution Solution 6
8. ere with downstream applications and thus must be washed from the oe well Solution column o Ensure that the dry spin under the Column Wash applications Ehana eanvever procedure is performed in order to remove traces of y ethanol prior to elution Ethanol is known to interfere with many downstream applications Perform RNAse free DNasel digestion on the RNA Genomic Larog amounts of sample after elution to remove genomic DNA DNA g contamination It is recommended that Norgen s contamination starting material used RNase Free DNase Kit Product 25710 be used for this step Related Products Product RNase Free DNase Kit 25710 Total RNA Purification Kit 17200 RNA Protein Purification Kit 23000 RNA DNA Protein Purification Kit 23500 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 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 2015 Norgen Biotek Corp P143400 3
9. ied Wash Solution prior to use Insufficient Genmmugalion Centrifuge the samples for at least 30 minutes or time and speed when longer at 4 000 x g or higher pelleting the stabilized g Poe nets blood RNA before column purification The RNA pellet was Pour off the supernatant slowly and carefully lost during decanting Insufficient Ensure that the appropriate amount of Lysis Buffer solubilization of blood was used Clogged Column Ensure that the centrifuge remains at room Centrifuge temperature throughout the procedure Temperatures temperature too low below 15 C may cause precipitates to form that can cause the columns to clog RNases may be introduced during the use of the kit RN s contaminati Ensure proper procedures are followed when working with RNA Please refer to Working with RNA at the beginning of this user guide RNA is DA en say In order to maintain the integrity of the RNA it is Degraded enough q y important that the procedure be performed quickly 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 Problem Possible Cause Solution and Explanation RNA does not RNA was not washed 3 times with the provided Wash Traces of salt from the binding step may remain in the sample if the column is not washed 3 times with Wash Solution Salt may interf
10. mn and centrifuge for 2 minute Discard the flowthrough Reassemble the spin column with its collection tube Apply 100 uL of the RNase free DNase solution prepared in Step 1 to the column and centrifuge at 14 000 x g 14 000 RPM for 1 minute Note Ensure that the entire DNase solution passes through the column If needed spin at 14 000 x g 14 000 RPM for an additional minute After the centrifugation in Step 4 pipette the flowthrough that is present in the collection tube back onto the top of the column Note Ensure Step 5 is performed in order to ensure maximum DNase activity and to obtain maximum yields of RNA in particular for small RNA species Incubate the column assembly at 25 30 C for 15 minutes Without any further centrifugation proceed directly to the second wash step in the Column Wash section Step 3c Troubleshooting Guide Problem Possible Cause Solution and Explanation Incomplete lysis of Ensure that the appropriate amount of Lysis Solution blood was used An alternative elution It is recommended that the Elution Solution supplied solution was used with this kit be used for maximum RNA recovery Ethanol was not Ensure that the appropriate amount of ethanol is added to the lysate added to the lysate before binding to the column Ethanol was not A Poor RNA added to the Wash Ensure that 50 mL of 95 ethanol is added to the Recovery Solution suppl
11. n 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 6 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 Protocol for Optional On Column DNA Removal Norgen s Preserved Blood RNA Purification Kit isolates total RNA with minimal amounts of genomic DNA contamination However an optional protocol is provided below for maximum removal of residual DNA that may affect sensitive downstream applications It is recommended that Norgen s RNase Free DNase Kit Product 25710 be used for this step 1 For every on column reaction to be performed prepare a mix of 15 uL of DNase I and 100 uL of Enzyme Incubation Buffer using Norgen s RNase Free DNase Kit Product 25710 Mix gently by inverting the tube a few times DO NOT VORTEX Note If using an alternative DNase I prepare a working stock of 0 25 Kunitz unit uL RNase free DNase solution according to the manufacturer s instructions A 100 uL aliquot is required for each column to be treated Perform the appropriate Preserved Blood RNA Isolation Procedure for your preserved blood sample up to and including Binding to Column Steps 1 and 2 of protocol Apply 400 uL of Wash Solution to the colu
12. ration from Tempus Blood RNA Tubes 1 Lysate Preparation from Tempus Blood RNA Tubes a Pour the entire contents of the Tempus tube into a new 50 mL conical tube b Add3 mL of Tempus Blood RNA Tube Diluent or enough to adjust the final volume to 12 mL Close the tube tightly and mix by vortexing vigorously for 30 seconds c Centrifuge the tube at 4 C at 3000 5000 x g minimum 4500 rpm on a Beckman JB 6 or equivalent swing bucket centrifuge for 30 minutes d Discard supernatant Note that the RNA pellet is transparent and invisible e Leave the tube inverted on paper towel for 2 minutes to dry off excessive liquid f Add 600 uL of Lysis Solution to the RNA pellet Vortex the tube for a few seconds to resuspend the pellet Add 300 uL of 95 100 ethanol provided by the user Vortex briefly to mix Proceed to Step 2 Binding RNA to Column ze 2 Binding RNA to Column a Assemble a column with one of the provided collection tubes b Apply up to 600 uL of the lysate with the ethanol from Step 1 onto the column and centrifuge for 1 minute at 2 3 500 x g 6 000 RPM 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 at 14 000 x g 14 000 RPM c Discard the flowthrough Reassemble the spin column with its collection tube d Repeat Step 2b and 2c as necessary Optional Step Norg
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