CST Genomic DNA Purification Kits -- Tissues
Contents
1. Observation Cause Solution RNA eluate is Magnetic beads Remove any magnetic beads using a discolored present in the magnetic separator MagnaRack is eluate available from Invitrogen see page 24 or centrifuge the sample in a microcentrifuge for 1 minute at maximum speed RNA is RNA e Use RNase free pipette tips with degraded contaminated aerosol barriers with RNase e Change gloves frequently e Swipe automatic pipettes with RNase AWAY solution Improper e If not processed immediately handling of quick freeze tissue immediately after sample from harvesting and store at 80 C or in harvest until liquid nitrogen lysis e Frozen samples must remain frozen until Lysis Buffer or TRIzol Reagent is added e Perform the lysis quickly after adding Lysis Buffer or TRIzol Reagent DNA DNase I not Be sure to add a tube containing 1 uL contamination added during DNase I supplied with iPrep PureLink the purification Total RNA Kit in position 11 of the iPrep protocol RNA Cartridge to perform DNase I digestion during the automated purification protocol 23 Appendix Accessory Products Additional Products TM The table below lists additional products available from Invitrogen that may be used with the iPrep RNA Kits For more information visit www invitrogen com or contact Technical Support page 25 Product A2. Mammalian Use the protocol below to prepare up to 50 mg of frozen or Tissue Lysate fresh tissue lysates 1 Place up to 50 mg of freshly minced mammalian tissue or frozen tissue into a sterile tube placed on ice Add 1 mL TRIzol Reagent supplied with the kit Ensure the tissue is completely immersed in the buffer Homogenize the tissue for a minimum of 1 minute using a hand held rotor stator tissue homogenizer Incubate the samples at room temperature for 5 minutes Add 0 2 mL chloroform and shake the tube vigorously by hand for 15 seconds Avoid vortexing the sample Incubate at room temperature for 2 3 minutes Centrifuge the sample at 12 000 x g for 15 minutes at 4 C After centrifugation the mixture separates into a lower red phenol chloroform phase an interphase and a colorless upper aqueous phase The volume of aqueous phase is 600 uL Transfer 400 500 uL of the colorless upper phase containing RNA to an iPrep Sample Tube and proceed to Isolating Total RNA using the iPrep Instrument page 17 Mammalian Procedure to prepare lysate from mammalian cells is Cells Lysate described below 1 For adherent cells up to 1 x 107 cells remove the growth medium from the culture plate For suspension cells up to 1 x 10 cells harvest the cells and centrifuge the cells at 250 x g for 5 minutes to pellet cells Remove the growth medium Resuspend the pelleted cells using 1 mL TRIzol Reagent Homogen
3. Add 0 2 mL chloroform and shake the tube vigorously by hand for 15 seconds Avoid vortexing the sample Incubate at room temperature for 2 3 minutes Centrifuge the sample at 12 000 x g for 15 minutes at 4 C After centrifugation the mixture separates into a lower red phenol chloroform phase an interphase and a colorless upper aqueous phase The volume of aqueous phase is 600 pL Transfer 400 500 uL of the colorless upper phase containing RNA to an iPrep Sample Tube and proceed to Isolating Total RNA using the iPrep Instrument page 17 Continued on next page 10 Using the iPrep TRIzol Plus Kit to Prepare Lysates Continued E coli Lysate 11 Procedure to prepare E coli cell lysate is described below 1 Harvest log phase bacteria 1 mL of overnight culture with ODeoo up to 2 0 or up to 5 x 10 cells by centrifugation at 500 x g for 5 minutes at 4 C If you are using a frozen cell pellet proceed directly to Step 2 2 Resuspend the cell pellet in 1 mL TRIzol Reagent and homogenize using a rotor stator homogenizer at maximum speed for at least 45 seconds 3 Add 0 2 mL chloroform and shake the tube vigorously by hand for 15 seconds Avoid vortexing the sample Incubate at room temperature for 2 3 minutes 5 Centrifuge the sample at 12 000 x 2 for 15 minutes at 4 C 6 After centrifugation the mixture separates into a lower red phenol chloroform phase an interphase and a colorl
4. Expected Examples of expected yield and purity are given as mean Yield and values with the range of values in parentheses Purity Total RNA was purified using the iPrep RNA Kits and the iPrep Purification Instrument using different samples RNA was eluted in 100 uL elution buffer yield was determined using the Quant iT RNA Assay Kit The UV absorbance ratios were measured using a NanoDrop ND 1000 spectrophotometer Sample iPrep TRIzol Plus RNA iPrep PureLink Total Kit RNA Kit Yield ug A260 A280 Yield ug A260 A280 293 cells 52 2 50 1 55 5 2 0 10 9 10 1 11 6 2 0 1 x 107 1 x 10 Liver 10 mg 44 2 43 3 46 3 2 1 40 8 35 9 44 5 2 1 Heart 10 mg 4 8 3 7 6 4 1 9 1 9 1 1 2 3 2 0 Kidney 10 mg 9 2 7 2 12 3 1 9 7 0 5 8 8 7 2 1 E coli 0 5 mL 80 3 79 0 81 7 1 9 64 8 58 0 68 5 2 0 culture ODeoo 1 Tomato leaf 23 9 23 1 25 4 2 0 kit not recommended for 100 mg this sample type 21 Troubleshooting Introduction Refer to the table below to troubleshoot problems with the TM kit To troubleshoot problems with the iPrep Purification Instrument refer to the manual supplied with the instrument Observation Cause Solution Low RNA Incomplete lysis e Perform lysis and homogenization as yield and recommended for each sample type using homogenization the appropriate lysis buffer as described on pages 8 12 e Decrease the amount of starting ma
5. 1 Prepare samples as described on page 8 for iPrep TRIzol Protocol Plus RNA Kit and page 12 for iPrep PureLink Total RNA Kit 2 Open the iPrep Card Slot and insert the iPrep Card Total RNA in the slot arrow on the card is at the top and card label is facing your left side 3 Turn ON the iPrep Instrument using the power switch on the left side of the instrument The digital display shows the version for the iPrep instrument which changes in few seconds to display the Main menu Press Start to run a protocol Open the iPrep instrument door and remove iPrep Racks to set up the platform 6 Remove the iPrep PureLink Total RNA Cartridges from the box To collect any solution from the foil tap the cartridge to deposit the solution at the bottom of the tube 7 Ifyou are performing DNase I digestion available with iPrep PureLink Total RNA Kit only insert one iPrep Sample and Elution Tube containing 1 uL DNase I in position 11 see page 3 without caps for each of the iPrep RNA Cartridge that is used 8 Load the desired number of cartridges on the iPrep Cartridge Rack Insert the loaded iPrep Cartridge rack on TM to the iPrep Platform Continued on next page Experienced Users Procedure continued Step Procedure Purification 9 Load the iPrep Tip and Tube Rack as follows Froroeol e Load the first row labeled as E with 1 13 elution
6. in few seconds to display the Main menu Press Start to run a protocol 5 Open the iPrep instrument door Remove the iPrep Cartridge Rack and iPrep Tip and Tube Rack to set up the platform 6 Remove the desired number of iPrep PureLink Total RNA Cartridges from the box To collect any solution from the foil tap the cartridge to deposit the solution at the bottom of the tube Note You can load 1 13 cartridges on the rack depending on the number of samples that you wish to process If you are loading less than 13 cartridges ensure that the remaining plastic ware tips and tubes are also loaded in the same order as the cartridges Continued on next page Isolating Total RNA Continued Purification Procedure continued from previous page Protocol 7 Ifyou are performing DNase I digestion available with Continued iPrep PureLink Total RNA Kit only for your samples insert one iPrep Sample and Elution Tube containing DNase I without caps in position 11 for each of the iPrep RNA Cartridge that is used 8 Load the cartridges on the iPrep Cartridge Rack and insert the loaded rack on the iPrep platform 9 Load the iPrep Tip and Tube Rack as follows see figure below e Load the first row labeled as E with 1 13 elution tubes without caps you may place the caps on the rack as shown in the figure below e Keep the second row labeled as T1 empty e Load the third row labeled as T2
7. to more purified material Always use proper microbiological aseptic techniques when working with RNA Use RNase AWAY Reagent page 24 to remove RNase contamination from work surfaces Continued on next page General Information Continued DNase Digestion Safety Information Follow the recommendations below to obtain the best results e Do not freeze the beads as this irreparably damages them Store the beads at room temperature e When using beads from the Reaction Cartridges collect any solution from the foil by tapping the cartridge to deposit the solution at the bottom of the tube Do not allow the beads to dry out as this renders them non functional TM e Discard Reaction Cartridges iPrep Tips and iPrep Tip Holders after use Do not reuse The option to perform DNase I digestion is available with iPrep PureLink Total RNA Kits only The DNase I digestion is performed during the purification protocol on the iPrep Instrument Place a tube containing 1 uL DNase I into the tube position of the cartridge position 11 page 3 During the automated purification protocol the DNase I Buffer included in the cartridge is added to the tube containing DNase I mixed well and DNase I is then added to the samples Appropriate incubation time is included in the automated protocol to perform DNase I digestion Note There is no need to perform the optional DNase I digestion step with the iPrep TR
8. with iPrep Tips in the iPrep Tip Holders e Load the fourth row labeled as S with sample tubes without caps containing samples Position 11 Row S Row T2 Row T1 Row E E Area to place caps om FA SK a 9s P A as 2 10 Read the sample and elution tube barcode if needed 11 Insert the iPrep Tip and Tube rack on the iPrep platform as shown above 12 Close the iPrep instrument door Continued on next page 18 Isolating Total RNA Continued Purification Procedure continued from previous page Protocol 13 Continued 14 15 16 17 18 19 20 2l Press Enter to continue Select the appropriate protocol followed by elution volume on the display when prompted Ensure that you have loaded the cartridges tubes and tips in the appropriate positions and elution tubes do not have any caps Make sure you have loaded a tube containing the sample in the heated tube position position 11 of the cartridge Press Start The automated purification protocol begins and various steps of the protocol including the approximate time remaining are displayed on the digital display Important Do not open the door once the protocol has begun To pause the protocol press the Stop key To resume the protocol after a pause press the Start key To cancel stop the protocol press the Stop key twice For details see the iPrep Instrument manual At the end of the run t
9. 52 purifications Reagents Amount iPrep PureLink Total RNA Cartridge Kit 1 kit iPrep Sample and Elution Tubes 3 x 52 tubes iPrep Tips and Tip Holders 1 bag with 52 tips and holders Box 2 The components supplied in iPrep PureLink Total RNA Contents Kit Box 2 are listed below Sufficient reagents are supplied to perform 52 purifications Reagents Amount Lysis Buffer 32mL RBC Lysis Buffer 400 mL Box 3 The iPrep PureLink Total RNA Kit Box 3 includes 52 uL Contents of DNase I 10 U uL in 20 mM sodium acetate pH 6 5 5 mM calcium chloride 50 glycerol and 0 1 mM PMSF Sufficient DNase I is supplied to perform 52 purifications TRizol The TRIzol Reagent 100 mL is supplied with the iPrep Reagent TRIzol Plus RNA Kit only Intended Use Sufficient TRIzol Reagent is supplied to perform 52 purifications For research use only Not intended for any animal or human therapeutic or diagnostic use vii Introduction Product Overview Introduction iPrep Purification Instrument System Overview The iPrep PureLink Total RNA and TRIzol Plus RNA Kits iPrep RNA Kits allow rapid and automated extraction of total RNA from a variety of samples including tissue plant and animal cells mammalian and bacterial cells and fresh whole blood iPrep TRIzol Plus RNA Kit only Total RNA is extracted from samples using the Dynabeads MyOne SILANE and iPrep Purification Instrument w
10. Izol Plus RNA Kit as we have observed minimal DNA contamination using this kit Follow the safety guidelines below when using the iPrep RNA Kits e Treat all reagents supplied in the kit as potential irritants e Always wear a suitable lab coat disposable gloves and protective goggles when handling whole blood samples e Dispose of blood samples as biohazardous waste Using the iPrep TRIzol Plus Kit to Prepare Lysates Introduction Instructions for preparing lysates from mammalian cells and tissues plant tissues and bacterial cells using the buffers included with the iPrep TRIzol Plus RNA Kit are described below See page 12 to prepare lysates using the iPrep PureLink Total RNA Kit To obtain high quality RNA follow the guidelines recommended on page 6 Materials Needed Samples for RNA isolation see page 6 for starting amounts Chloroform Rotor stator homogenizer Liquid nitrogen and mortar and pestle for plant tissues Components Supplied with the Kit TRIzol Reagent Note Maintain frozen tissue at 80 C prior to lysis Cool tubes in dry ice before placing frozen tissue in them Thawing of frozen tissue prior to lysis may result in RNA degradation and loss of RNA yield Fast and complete disruption of tissue during the lysis step is important to prevent RNA degradation Continued on next page Using the iPrep TRIzol Plus Kit to Prepare Lysates Continued
11. c beads are separated from the lysate using magnetic separation The beads are thoroughly washed with Wash Buffers to remove contaminants The total RNA is then eluted in Elution Buffer Continued on next page Product Overview Continued Advantages System Specifications The iPrep RNA Kits provide the following advantages Uses a magnetic bead based technology to isolate total RNA without the need for centrifugation or vacuum manifolds Rapid and automated purification of total RNA within 30 45 minutes from a wide range of samples including difficult tissue samples such as fatty and fibrous tissue Purifies fully representative RNA includes high and low molecular weight RNA molecules Pre filled reagent cartridges provide easy set up and consistent results Minimal contamination with DNA Purified RNA demonstrates improved downstream performance in applications such as microarray analysis or real time quantitative RT PCR qRT PCR Starting Material See page 6 for sample amount Bead Size 1 um Bead Amount per Reaction 2 4 mg Number of Samples Up to 13 Elution Volume 50 uL or 100 uL RNA Yield Varies see page 21 The RNA yield depends on the sample type and quality iPrep Purification Instrument Introduction iPrep Reaction Cartridge TM The iPrep Purification Instrument is a benchtop automated nucleic acid purification instrument with integrated Magnetic and Syringe Unit ca
12. e 18 While assembling the tips on the rack insert the iPrep Tips into the iPrep Tip Holders using gloved hands Always use the tips with the holders to prevent any contamination Tip Specifications Tip Material Polypropylene with filter barriers Tip Holder Material Polypropylene Volume 5 1000 pL Tip Dimension 3 9 inches 1 x 0 43 inches d iPrep Tip Holder iPrep Tip Two sets of iPrep Tubes are required for the purification protocol The iPrep Sample and Elution Tubes are included with each iPrep Kit and placed on the iPrep Tip and Tube Rack as described on page 18 Tube Specifications Material Polypropylene Capacity 1 5 mL Style Tubes with caps Dimensions 1 7 inches 1 x 0 4 inches d Continued on next page iPrep Purification Instrument Continued iPrep Card Total RNA iPrep Platform TM To use the iPrep RNA Kits with the iPrep Purification Instrument you need to purchase the iPrep Card Total RNA page 24 The iPrep Card Total RNA is pre programmed with the purification protocol for RNA that directs the volume of reagents used and incubation time Always store the card in the box protected from light To avoid damaging the card e Donot drop or bend the card e Do not wipe or clean the card using volatile chemicals such as alcohol or equivalent e Do not expose the card to water The platform on the iPrep Instrument allows the place
13. e box Kit Contents and Storage Types of This manual is supplied with the following kits Manuals Product Quantity Cat no iPrep PureLink Total RNA Kit 1 kit IS 10006 iPrep TRIzol Plus RNA Kit 1 kit IS 10007 Kit The table below shows the components supplied with Components iPrep PureLink Total RNA and TRIzol Plus RNA Kits iPrep RNA Kits Components iPrep PureLink iPrep TRIzol Total RNA Kit Plus RNA Kit iPrep Total RNA Box 1 Y V iPrep PureLink Total RNA Box 2 Y iPrep PureLink Total RNA Box 3 Y TRIzol Reagent y Shipping and The iPrep PureLink Total RNA and TRIzol Plus RNA Storage Kits iPrep RNA Kits are shipped as described below Upon receipt store each component as described below All components are guaranteed stable for 6 months when stored properly Item Shipping Storage iPrep Total RNA Box 1 Room temperature Room temperature iPrep PureLink Total RNA Box 2 Room temperature Room temperature iPrep PureLink Total RNA Box 3 Dry ice 20 C TRIzol Reagent Room temperature Room temperature vi Continued on next page Kit Contents and Storage Continued Box 1 The components supplied in iPrep RNA Kits Box 1 are Contents listed below Sufficient reagents are supplied to perform
14. ess upper aqueous phase The volume of aqueous phase is 600 uL 7 Transfer 400 500 uL of the colorless upper phase containing RNA to an iPrep Sample Tube and proceed TM to Isolating Total RNA using the iPrep Instrument page 17 Using the iPrep PureLink Kit to Prepare Lysates Introduction Materials Needed Note Instructions for preparing lysates from mammalian cells and tissues whole blood and bacterial cells using the buffers included with the iPrep PureLink Total RNA Kit are described below See page 8 to prepare lysates using the iPrep TRIzol Plus RNA Kit To obtain high quality RNA follow the guidelines recommended on page 6 e Samples for RNA isolation see page 6 for starting amounts e mercaptoethanol e Rotor stator homogenizer e 10 SDS and 10 mg mL lysozyme solution in TE for bacterial samples e Optional Homogenizer page 24 for tissue samples Components Supplied with the Kit e Lysis Buffer RBC Lysis Buffer e Maintain frozen tissue at 80 C prior to lysis Cool tubes in dry ice before placing frozen tissue in them Thawing of frozen tissue prior to lysis may result in RNA degradation and loss of RNA yield e For samples that are difficult to lyse use TRIzol Reagent as described on page 8 e Fast and complete disruption of tissue during the lysis step is important to prevent RNA degradation Continued on next page 12 Using the iPrep PureL
15. he instrument beeps briefly and the digital display shows Protocol Finished for 10 seconds The Main menu appears after 10 seconds Open the instrument door Remove and cap the elution tubes containing the purified nucleic acid Store the purified RNA as described below Discard the used cartridges tips and sample tubes into biohazard waste Do not reuse the cartridges To purify more samples using the same iPrep Card load the racks with new cartridges tips and samples and start the protocol as described above If you are not using the instrument close the instrument door and turn the power switch to OFF Note Remove the iPrep Card and store the card in the box protected from light if you are planning to use the instrument with a different iPrep Card If the instrument is repeatedly used with the same iPrep Card you can keep the card in the instrument Storing RNA Store the RNA on ice if you will use the RNA within a few hours for the desired downstream application Aliquot purified RNA and store at 80 C Avoid repeated freezing and thawing 19 RNA Quantitation and Analysis RNA Yield Analyzing RNA Quality Total RNA is easily quantitated using the Quant iT RNA Kits or UV absorbance at 260 nm Quant iT RNA Kits The Quant iI RNA Kit page 24 provides a rapid sensitive and specific method for RNA quantitation with minimal interference from DNA protein or other common contami
16. ink Kit to Prepare Lysates Continued Mammalian Tissue Lysate Mammalian Cells Lysate 13 Use the protocol below to prepare up to 10 mg of frozen or fresh tissue lysates 1 Place up to 10 mg of freshly minced mammalian tissue or frozen tissue into a sterile tube placed on ice Add 600 pL Lysis Buffer supplied with the kit Ensure the tissue is completely immersed in the buffer Homogenize the tissue for a minimum of 1 minute using a hand held rotor stator tissue homogenizer Centrifuge the lysate at 12 000 x g for 5 minutes at room temperature to remove any particulate material Note If the lysate is viscous or contains cell debris clarify the lysate using the Homogenizer available from Invitrogen page 24 Transfer the supernatant to an iPrep Sample Tube and proceed to Isolating Total RNA using the iPrep Instrument page 17 Procedure to prepare lysate from mammalian cells is described below T For adherent cells up to 1 x 10 cells remove the growth medium from the culture plate For suspension cells up to 1 x 10 cells harvest the cells and centrifuge the cells at 250 x g for 5 minutes to pellet cells Remove the growth medium Resuspend cells in 600 uL Lysis Buffer supplied with the kit Mix well by vortexing or pipetting up and down until the cells appear lysed Homogenize the sample for a minimum of 2 minutes using a hand held rotor stator tissue homogenizer TM Transfer the cell l
17. invitrogen by technologies iPrep PureLink Total RNA and TRIzol Plus RNA Kits For purification of total RNA from tissue cells and blood using the iPrep Purification Instrument Catalog nos IS 10006 IS 10007 Rev date 18 November 2010 Manual part no 100001249 MAN0000404 Contents Experienced Users Procedure sse e eene iv Kit Coritents and Storage cette iet edet ee vi Introduclion inse eee to dede mete cance atta adeeb acd vo O NNUS 1 Product Overview iPrep Purification Instrument sseeeeeeeeenene eet 3 Mth il EDD 6 General Information RE ERE tees 6 Using the iPrep TRIzol Plus Kit to Prepare Lysates sss 8 Using the iPrep PureLink Kit to Prepare Lysates sss 12 Isolating Total RNA ire tice ette reete t ee ia RNA Quantitation and Analysis Expected Results ee notet tenter ted ied tee ine Troubl shootitig e tete tree tenete tede ei 22 Appendgdlbx 5 eene aceasta DIM e ere eue re tae tas 24 Accessory Product eintreten tere pecie pe eren 24 Technical Suppott rette bett ertet eet 25 Purchaser Notification ice rete te iste erste th to eee RE ERA 27 Experienced Users Procedure Introduction This quick reference sheet is included for experienced users of the iPrep PureLink Total RNA and iPrep TRIzol Plus RNA Kits For more details refer to this manual Step Procedure Purification
18. ithin 30 45 minutes without the use of centrifugation The purified total RNA is suitable for use in sensitive downstream applications including gene expression studies such as microarray analysis or real time quantitative RT PCR qRT PCR The iPrep RNA Kits are designed for use with the iPrep Purification Instrument The iPrep Purification Instrument is a benchtop automated nucleic acid purification instrument with integrated Magnetic and Syringe Unit capable of purifying nucleic acids from up to 13 samples 12 samples 1 positive control using a magnetic bead based technology See page 3 for details on the iPrep Purification Instrument The iPrep RNA Kits combine the sensitivity and capacity of Dynabeads MyOne SILANE with the speed and convenience of the iPrep Instrument to allow automated purification of high quality RNA from up to 13 samples 12 samples 1 positive control within 30 45 minutes The Dynabeads MyOne SILANE are monodisperse magnetic beads 1 um with an optimized silica like surface chemistry and a high specific surface area Purification is achieved using a simple magnetic bead based purification procedure and avoids the use centrifuges or vacuum manifolds Samples are lysed manually using the Lysis Buffer RBC Lysis Buffer or TRIzol Reagent based on sample type The lysate is mixed with Dynabeads MyOne SILANE for subsequent RNA binding to the beads The RNA bound magneti
19. ize the sample for a minimum of 1 minute using a hand held rotor stator tissue homogenizer Incubate at room temperature for 5 minutes Add 0 2 mL chloroform and shake the tube vigorously by hand for 15 seconds Avoid vortexing the sample Continued on next page Using the iPrep TRIzol Plus Kit to Prepare Lysates Continued Mammalian 6 Cells Lysate 7 Continued 8 9 Incubate at room temperature for 2 3 minutes Centrifuge the sample at 12 000 x g for 15 minutes at 4 C After centrifugation the mixture separates into a lower red phenol chloroform phase an interphase and a colorless upper aqueous phase The volume of aqueous phase is 600 uL Transfer 400 500 uL of the colorless upper phase containing RNA to an iPrep Sample Tube and proceed to Isolating Total RNA using the iPrep Instrument page 17 Plant Lysate Procedure to prepare plant tissue lysate is described below 1 2 10 11 Place up to 100 mg of plant tissue in a pre chilled mortar Immediately add liquid nitrogen and grind the tissue to a fine powder Do not allow the liquid nitrogen to completely evaporate Transfer the ground powder with remaining liquid nitrogen to a fresh RNase free tube Place the tube on dry ice and allow the liquid nitrogen to evaporate Add 1 mL TRIzol Reagent and homogenize the sample for 2 minutes using a hand held rotor stator homogenizer Incubate at room temperature for 5 minutes
20. lations citations handbooks etc e Complete technical support contact information e Access to the Invitrogen Online Catalog e Additional product information and special offers Contact Us For more information or technical assistance call write fax or email Additional international offices are listed on our Web page www invitrogen com Corporate Headquarters European Headquarters 5791 Van Allen Way Inchinnan Business Park Carlsbad CA 92008 USA 3 Fountain Drive Tel 1 760 603 7200 Paisley PA4 9RF UK Tel Toll Free 1 800 955 6288 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Tech Fax 44 0 141 814 6117 E mail E mail tech_support invitrogen com eurotech invitrogen com Continued on next page 25 Technical Support Continued SDS Requests Limited Warranty SDSs Safety Data Sheets are available on our website at www invitrogen com sds Invitrogen is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 10076 satisfied with our products and our service If you should have any questions or concerns about an Invitrogen product or service contact our Technical Support Representatives Invitrogen warrants that all of its products will perform according to specifications stated on the certificate of analysis The company will replace free of charge any product that does not meet those specifications This warranty limits In
21. ment of iPrep Tip and Tube Rack and iPrep Cartridge Rack that are filled with plastic disposables and reagent cartridges required for the purification protocol Set up the platform as shown in the figure on page 18 for the iPrep RNA Kits Methods General Information User Supplied In addition to the reagents supplied with the kit you also Materials need the following materials and instrumentation e iPrep Purification Instrument page 24 e iPrep Card Total RNA page 24 e Samples see below Starting The various sample types and amounts that can be Material processed using the system are listed in the table below Sample type iPrep TRIzol Plus iPrep PureLink RNA Kit RNA Kit Animal tissue Up to 50 mg Up to 10 mg Mammalian cells Up to 1 x 10 cells Up to 1 x 10 cells Plant tissue 100 mg Not recommended Fresh whole blood Not recommended Up to 1 0 mL Bacterial cells Up to 5 x 10 Up to 1 x 10 Guidelines for Handling RNA Follow the guidelines below to prevent RNase contamination and to maximize the RNA yield Use disposable individually wrapped sterile plastic ware Use only sterile disposable RNase free pipette tips and microcentrifuge tubes Wear disposable gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin change gloves frequently particularly as the protocol progresses from crude extracts
22. mount Cat no iPrep Purification Instrument 1 unit IS 10000 iPrep Card Total RNA 1 card IS 10014 iPrep Card gDNA Blood 1 card IS 10012 iPrep Card gDNA Tissue 1 card IS 10013 iPrep Card gDNA Forensic includes 1 card IS 10011 buccal protocol iPrep PureLink gDNA Blood Kit 1 kit IS 10005 iPrep ChargeSwitch Forensic Kit 1 kit 52 purifications IS 10002 iPrep ChargeSwitch Buccal Cell Kit 1 kit 52 purifications IS 10003 iPrep ChargeSwitch gDNA Tissue Kit 1 kit 52 purifications IS 10004 iPrep Tip and Tube Rack 1 rack IS 10101 iPrep Cartridge Rack 1 rack IS 10102 Quant iT RNA Assay Kit 1000 assays 1 kit Q33140 5 100 ng Qubit Fluorometer 1 each Q32857 MagnaRack Magnetic Separator 1 rack CS15000 Homogenizer 1 pack of 50 12183 026 RNase AWAY Reagent 250 mL 10328 011 E Gel E Gel Agarose Gels are bufferless pre cast agarose gels Agarose Gels designed for fast convenient electrophoresis of DNA and DNA samples E Gel agarose gels are available in different Ladders agarose percentages and well formats In addition a large variety of DNA ladders is available from Invitrogen for sizing DNA For more information about these products see www invitrogen com or call Technical Support page 25 24 Technical Support World Wide Visit the Invitrogen website at www invitrogen com for Web e Technical resources including manuals vector maps and sequences application notes SDSs FAQs formu
23. nants that affect UV absorbance readings The kits contain a state of the art quantitation reagent and pre diluted standards for standard curve The assay is performed in a microtiter plate format and is designed for reading in standard fluorescent microplate readers fluorometers UV Absorbance To determine the quantity by UV absorbance 1 Dilute an aliquot of the total RNA sample in 10 mM Tris HCl pH 7 5 Mix well Transfer to a cuvette 1 cm path length Note The RNA must be in a neutral pH buffer to accurately measure the UV absorbance 2 Determine the OD o of the solution using a spectrophotometer blanked against 10 mM Tris HCl pH 7 5 3 Calculate the amount of total RNA using the following formula Total RNA ug A260 x 40 pg A20 x 1 mL x dilution factor x total sample volume mL Typically total RNA isolated using the iPrep RNA Kits has an OD260 280 of 71 8 when samples are diluted in Tris HCl pH 7 5 An OD260 280 of 71 8 indicates that RNA is reasonably clean of proteins and other UV chromophores that could either interfere with downstream applications or negatively affect the stability of the stored RNA RNA quality may also be assessed using a bioanalyzer Agarose gel electrophoresis of RNA isolated using the iPrep RNA Kits show the 285 to 185 band ratio to be gt 1 5 RNA is judged to be intact if discreet 285 and 18S ribosomal RNA bands are observed 20 Expected Results
24. omponents is conveyed expressly by implication or by estop pel This product is for internal research purposes only and is not for use in commercial applications of any kind including without limitation quality control and commercial services such as reporting the results of purchaser s activities for a fee or other form of consideration For information on obtaining additional rights please contact outlicensing lifetech com or Out Licensing Life Technologies 5791 Van Allen Way Carls bad California 92008 2010 Life Technologies Corporation All rights reserved The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners TRIzol is a trademark of Molecular Research Center Inc NanoDrop is a registered trademark of NanoDrop Technologies LLC RNase Away is a trademark of Molecular Bio Products Inc invitrogen by Life technologies Corporate Headquarters 5791 Van Allen Way Carlsbad CA 92008 T 1 760 603 7200 F 1 760 602 6500 E tech_support invitrogen com For country specific contact information visit our web site at www invitrogen com
25. p Purification Instrument page 24 e iPrep Card Total RNA page 24 Components Supplied with the Kit e iPrep PureLink RNA Cartridge Kit e iPrep Sample and Elution Tubes e iPrep Tips e iPrep Tip Holders e DNase I optional Perform the following before starting e Prepare lysates as described on pages 8 12 e Ensure that you have the iPrep Card Total RNA page 24 to run the protocol e Make sure the iPrep Purification Instrument is unpacked and installed Continued on next page 16 Isolating Total RNA Continued Purification Protocol 17 Purify total RNA using the iPrep Purification Instrument as described below TM For details on using the iPrep Purification Instrument refer to the manual supplied with the instrument Insert the iPrep Card Total RNA available separately from Invitrogen page 24 prior to turning on the instrument 1 Ensurethe power switch on the iPrep Instrument is on the OFF position 2 Open the iPrep Card Slot and insert the iPrep Card Total RNA into the slot in the correct orientation arrow on the card is at the top and card label is facing your left side 3 Using the power switch located on the left side of the instrument turn ON the instrument If the card is fully inserted in the correct orientation all axes return to their original positions automatically The digital display shows the version for the iPrep which changes
26. pable of purifying nucleic acids from up to 12 samples and one positive control Each iPrep Instrument consists of the Magnetic and Syringe Unit and a platform A pre programmed iPrep Protocol Card controls the purification parameters such as buffer volumes mixing steps and incubation time For more details on the iPrep Purification Instrument see the manual supplied with the instrument The iPrep Reaction Cartridges are supplied with iPrep Kits and are designed to fit onto the iPrep Cartridge Rack in only one orientation Each cartridge is pre filled with reagents required for the iPrep RNA protocol Each cartridge has 12 positions with 10 sealed wells and two heating positions position 12 with an empty well and position 11 to add an empty or reagent filled tube For the iPrep RNA Kits positions 1 10 contain wells filled with reagents Cartridge Specifications Material Polypropylene cartridge sealed with laminated aluminum foil Max Volume 1000 uL well Dimension 5 9 inches 1 x 1 2 inches w x 0 7 inches d Note The image below shows an example of an iPrep Reagent Cartridge and is not an image of an iPrep RNA Cartridge Position 11 Continued on next page iPrep Purification Instrument Continued iPrep Tips and Tip Holders iPrep Tubes The iPrep Tips and Tip Holders are included with the iPrep Kits and are placed on the iPrep Tip and Tube Rack as described on pag
27. terial e Cut tissue samples into smaller pieces and ensure the tissue is completely immersed in buffer to achieve optimal lysis Poor quality of starting material e The yield and quality of RNA isolated depends on the type and age of the starting material e Be sure to use fresh sample and process immediately after collection or freeze the sample at 80 C or in liquid nitrogen immediately after harvesting Insufficient amount of Dynabeads MyOne SILANE added During shipping some Dynabeads MyOne SILANE solution may adhere to the sealing foil of the cartridge To collect any bead solution from the foil tap the cartridge to deposit the bead solution at the bottom of the tube Used less than the recommended sample amount See page 6 for recommended starting amount for various sample types depending on the type of kit you are using No RNA Magnetic beads e Store cartridge containing the beads at room recovered stored or temperature Do not freeze the cartridge as handled the beads may be irreparably damaged improperly e Make sure the beads are in solution at all times and do not dry Dried beads are non functional Too much Decrease the amount of starting material use starting material See page 6 for recommended starting amount clogs tips Make sure the lysates is clear and does not contain particulate material Continued on next page 22 Troubleshooting Continued
28. the iPrep PureLink Kit to Prepare Lysates Continued E coli Lysate 15 Procedure to prepare E coli cell lysate is described below Prepare 100 uL TE buffer 10 mM Tris HCl 1 mM EDTA pH 8 0 containing 1 mg lysozyme enzyme Store on ice until use You will need 100 uL lysozyme solution sample Using the Lysis Buffer included with the kit prepare 500 uL Lysis Buffer containing 10 uL B mercaptoethanol per sample Harvest log phase bacteria 0 5 mL of overnight culture with ODeo up to 1 0 or up to 1 x 10 cells by centrifugation at 500 x g for 5 minutes at 4 C If you are using a frozen cell pellet proceed to Step 4 Resuspend the cell pellet in 100 uL TE buffer containing 1 mg lysozyme from Step 1 Add 0 5 uL 10 SDS to the lysate and mix well by vortexing Incubate at room temperature for 5 minutes Add 500 pL Lysis Buffer containing B mercaptoethanol Step 2 to the lysate Homogenize using a rotor stator homogenizer at maximum speed for at least 45 seconds Centrifuge the homogenate at 2 600 x g for 5 minutes at room temperature Transfer the lysate to an iPrep Sample Tube and proceed to Isolating Total RNA using the iPrep Instrument page 17 Isolating Total RNA Introduction Materials Needed Before Starting Instructions to isolate total RNA using the iPrep RNA Kits with the iPrep Purification Instrument are described below e Lysate prepared as described on pages 8 12 e iPre
29. tubes Continued 10 11 12 13 14 15 16 17 18 19 20 21 without caps e Keep the second row labeled as T1 empty e Load the third row labeled as T2 with iPrep Tips in the iPrep Tip Holders e Load the fourth row labeled as S with sample tubes without caps containing the lysate Read the sample and elution tube barcodes if needed Insert the iPrep Tip and Tube Rack on to the iPrep Platform Close the iPrep instrument door Press Enter J to continue Select the appropriate elution volume on the display Press Start The automated purification protocol begins and various steps of the protocol including the approximate time remaining are displayed on the digital display At the end of the run the instrument beeps briefly and the digital display shows Protocol Finished for 10 seconds The Main menu appears after 10 seconds Open the instrument door Remove and cap the elution tubes containing the purified nucleic acid Aliquot and store the purified RNA at 80 C Discard the used cartridges tips and tubes into biohazard waste Do not reuse the cartridges To purify more samples using the same iPrep Card load the racks with new cartridges tips tubes and samples and start the protocol as described If you are not using the instrument close the instrument door and turn the power switch to OFF Optional Remove the iPrep Card and store card in th
30. vitrogen Corporation s liability only to the cost of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instructions Invitrogen reserves the right to select the method s used to analyze a product unless Invitrogen agrees to a specified method in writing prior to acceptance of the order Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore Invitrogen makes no warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications please report it to our Technical Support Representatives Invitrogen assumes no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose 26 Purchaser Notification Limited Use Label License No 358 Re search Use Only 27 The purchase of this product conveys to the purchaser the limited non transferable right to use the purchased amount of the product only to perform internal research for the sole benefit of the purchaser No right to resell this product or any of its c
31. ysate to an iPrep Sample Tube and proceed to Isolating Total RNA using the iPrep Instrument page 17 Continued on next page Using the iPrep PureLink Kit to Prepare Lysates Continued Whole Blood Procedure to prepare whole blood sample is described below Sample To isolate RNA from blood you need to first isolate the white Preparation blood cell WBC fraction and then lyse the WBC to prepare whole blood lysates 1 Toup to 1 mL of fresh whole blood sample add 5 volumes of RBC Lysis Buffer supplied with the kit For example To 1 mL of fresh whole blood add 5 mL RBC Lysis Buffer Incubate samples on ice for 10 minutes with intermittent vortexing every 3 minutes Centrifuge samples at 400 x g for 10 minutes at 4 C Remove and discard the supernatant Resuspend the pellet in 2 volumes of RBC Lysis Buffer Mix well by vortexing For example Resuspend the pellet in 2 mL for 1 mL starting volume of blood used RBC Lysis Buffer Centrifuge samples at 400 x g for 10 minutes at 4 C Remove and discard the supernatant Resuspend the resulting WBC pellet in 600 uL Lysis Buffer Homogenize the cells using a rotor stator homogenizer at maximum speed for at least 45 seconds Centrifuge the homogenate at 2 600 x g for 5 minutes at room temperature Transfer the lysate to an iPrep Sample Tube and proceed to Isolating Total RNA using the iPrep Instrument page 17 Continued on next page 14 Using
Download Pdf Manuals
Related Search