FastTrack® Kit - Thermo Fisher Scientific
Contents
1. Additional kit reagent is available separately from Invitrogen See the table below for ordering information A large variety of RNA and DNA purification kits is available from Invitrogen For details visit www invitrogen com naprep or contact Technical Support Item Amount Catalog no Oligo dT Cellulose 1 gram R545 01 Materials Shipping Storage Kit Contents Quick Reference Card The FastTrack 2 0 mRNA Isolation Kit is shipped at room temperature Upon receipt store the kit at room temperature for up to six months Remove the six tubes of premeasured oligo dT cellulose and store in a dessicator protected from light at room temperature as oligo dT cellulose is hydroscopic and sensitive to light For long term storage gt 6 months store the oligo dT cellulose at 20 C in a dessicator protected from light The Proteinase K solution is stable for 1 year when stored at room temperature For long term storage gt 1 year or if room temperature is gt 25 C store the Proteinase K solution at 4 C The FastTrack 2 0 mRNA Isolation Kit contents are listed below for K1593 02 6 reactions The kit for 18 reactions K1593 03 contains three times the amount of reagents listed in the table below Item Quantity Composition Stock Buffer 100 ml 200 mM NaCl 200 mM Tris HCl pH 7 5 1 5 mM MgCl 2 SDS Proteinase K 20 mg ml in 2x 1 25 ml2. 5 minutes at room temperature Remove the supernatant carefully from the resin bed Proceed to the washing procedure next page Continued on next page Basic mRNA Isolation Method Continued Washing Oligo dT Cellulose Elution and Precipitation of the mRNA 10 1 Resuspend the oligo dT cellulose in 20 ml Binding Buffer Centrifuge at 3 000 x g in a centrifuge for 5 minutes at room temperature Remove the Binding Buffer from the resin bed Resuspend the resin in 10 ml Binding Buffer and centrifuge as in Step 1 Remove the buffer from the resin bed Resuspend the resin in 10 ml Low Salt Wash Buffer and centrifuge as in Step 1 This low salt wash removes SDS and contaminating RNAs such as rRNAs Repeat Step 3 until the buffer no longer has any bubbles after centrifugation at least 2 4 times After the last wash resuspend the oligo dT cellulose in Low Salt Wash Buffer at a final volume of 800 ul Transfer the oligo dT cellulose into a spin column inside the spin column micro centrifuge set to perform the last washing steps Centrifuge at 5 000 x g for 10 seconds at room temperature Remove the column from microcentrifuge tube and decant the liquid inside the tube Repeat Steps 5 and 6 as many times as necessary 2 3 times to transfer the oligo dT to the spin column To wash place the spin column back into the tube fill the tube to the top with Low Salt Wash Buffer 500 ul and mix the buffer into the cellulose
3. Proprietary storage buffer Binding Buffer 2x 110 ml 500 mM NaCl 220 ml 10 mM Tris HCl pH 7 5 in DEPC treated water Low Salt Wash Buffer 3 x 100 ml 250 mM NaCl 300 ml 10 mM Tris HCl pH 7 5 in DEPC treated water Elution Buffer 2x8 ml 10 mM Tris HCl pH 7 5 in DEPC treated water 2 M Sodium Acetate 4 ml 2 M Sodium Acetate pH 5 2 in DEPC treated water 5 M NaCl 8 ml 5 M NaCl in DEPC treated water Pre measured Oligo dT 20 6 x 75 mg Lyophilized oligo dT cellulose 30 Cellulose Powder Microcentrifuge Tubes with 6 each Disposable Spin Columns Microcentrifuge Tubes 6 each Also included in this kit is a Quick Reference Card that can be used as a checklist Each step can be conveniently marked with a laboratory marker pen to keep track of steps Marks may be removed with ethanol before the next use Continued on next page Materials Continued Materials Supplied by User Solutions Phosphate Buffered Saline PBS to wash tissue culture cells see page 6 100 ethanol 70 ethanol Equipment Sterile 50 ml cell culture centrifuge tubes Water baths 45 C 65 C Liquid nitrogen tissues Sterile knife tissues 15 20 cc syringes with 18 21 gauge needles Motor driven homogenizer for tissues Tissuemizer or Omni Mixer Table top centrifuge Rocking platform or rotating wheel Mortar pestle and 50 ml Dounce Homogenizer tissues optional Methods Preparing Samples General Information
4. Cells or 0 4 1 gram Tissue Detergent lysis plus Proteinase K Batch binding with NaCI and oligo dT cellulose powder Low salt wash Spin column elution mRNA for cDNA Synthesis Oocyte Microinjection Northerns and RT PCR Continued on next page Overview Continued Benefits of the FastTrack Kit Types of Kits Micro FastTrack Kit Additional Products The FastTrack 2 0 mRNA Isolation Kit provides the following benefits Feature Benefit Complete kit with RNase free solutions Ensures success in your laboratory No total RNA isolation necessary Saves considerable time Pre measured high quality oligo dT cellulose powder Produces higher yields of mRNA over oligo dT tablets Efficient binding of mRNA to oligo dT cellulose Improves yield Minimal centrifugation manipulation Reduces mRNA degradation and loss The FastTrack 2 0 mRNA Isolation Kit is available in two sizes Item Reactions Catalog no FastTrack 2 0 Kit K1593 02 FastTrack 2 0 Kit 18 3 x 6 K1593 03 The Micro FastTrack mRNA Isolation Kit is also available from Invitrogen and allows efficient mRNA isolation from small samples 1 x 10 5 x 10 tissue culture cells 10 200 mg tissue or 100 ug total RNA Item Reactions Catalog no Micro FastTrack Kit K1520 02 Micro FastTrack Kit 60 3 x 20 K1520 03
5. Introduction General Handling of RNA Preparing Lysis Buffer ae 2 N D Y Sy A _ A je gt Review this section carefully before starting and familiarize yourself with the basic mRNA isolation protocol on page 9 The method of sample preparation depends on whether your sample is tissue culture cells tissues including plants or total RNA The lysate or total RNA solution you produce in this section is then used directly for mRNA isolation Once you lyse your sample do not stop until the mRNA is in ethanol see Elution and Precipitation of the mRNA Step 6 page 10 When working with RNA e Use disposable individually wrapped sterile plasticware e Use only sterile new pipette tips and microcentrifuge tubes e Glassware used for RNA work should be cleaned with detergent thoroughly rinsed autoclaved then oven baked at gt 210 C for at least three hours before use e Wear latex gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin e Always use proper microbiological aseptic technique when working with RNA You may use RNase AWAY Reagent a non toxic solution available from Invitrogen Catalog no 10328 011 to remove RNase contamination from surfaces For further information on controlling RNase contamination see Ausubel er al 1990 or Sambrook et al 1989 Prepare the FastTrack 2 0 Lysis Buffer immediately before Follow the instructions belo
6. Isolation Method Step 1 page 9 Preparing Samples Plants and Total RNA Plant Tissue MRNA Isolation from Total RNA Plant tissue may need to be ground with a mortar and pestle in liquid nitrogen then homogenized with a motor driven homogenizer to improve cell breakage 1 Harvest 1 2 g plant tissue and quickly freeze in liquid nitrogen 2 Place tissue in a mortar precooled with liquid nitrogen Cover the tissue with more liquid nitrogen and grind with a pestle until a fine powder is obtained Add more liquid nitrogen as needed to keep the tissue covered with liquid nitrogen while grinding 3 Transfer tissue and liquid nitrogen to a sterile 50 ml centrifuge tube As soon as liquid nitrogen evaporates add 15 ml FastTrack 2 0 Lysis Buffer with Proteinase K page 5 4 Quickly homogenize the tissue using a motor driven homogenizer or a Dounce homogenizer until a smooth homogenate with no visible particulate matter is obtained 15 30 seconds Keep foaming to a minimum by adjusting the speed 5 Proceed to the Basic mRNA Isolation Method Step 1 page 9 The 75 mg aliquot of oligo dT cellulose binds 75 ug polyA RNA Assuming total RNA is 1 5 polyA we recommend that you start with 1 0 mg of total RNA per mRNA isolation 1 Ethanol precipitate total RNA and wash with 80 ethanol 2 Resuspend the pellet in 100 ul of Elution Buffer 3 Add the RNA solution to 10 ml FastTrack 2 0 Lysis Buffer page 5 in a s
7. Low yields of mRNA Proteinase K depleted because large amounts of protein in the sample compete out RNase Reduce amount of starting material mRNA is not completely eluted off oligo dT cellulose Perform another set of elutions Heat the elution buffer to 65 C and pass over the column Ruptured membrane in spin column loss of oligo dT cellulose Be careful when resuspending oligo dT cellulose mRNA is contaminated with rRNA Poor removal of rRNA during Low Salt Wash Mix the Wash buffers gently into the oligo dT cellulose with the pipette tip Viscous lysate Sample too large mRNA will bind to oligo dT cellulose if the viscosity is reduced Add 15 ml more of FastTrack 2 0 Lysis Buffer and process as 2 samples Remember to use 2 spin columns and 2 aliquots of oligo dT cellulose Large amounts of DNA Split the sample as above and shear with a 21 gauge needle Each kit component is sterile and free of ribonuclease contamination and has been lot qualified for optimum performance in the FastTrack 2 0 mRNA Isolation Kit We do not recommend substituting your own reagents for any of the reagents supplied with the kit Technical Support World Wide Web Visit the Invitrogen website at www invitrogen com for e Technical resources including manuals vector maps and sequences application notes MSDSs FAQs formulations citations han
8. bed with a sterile pipette tip Centrifuge for 10 seconds Be careful not to damage the membrane as you will lose the resin and your sample Repeat Step 7 at least 3 times or until the OD 6 of the flow through is lt 0 05 Be sure to mix the buffer into the cellulose bed 10 Place the spin column into a new sterile and RNase free microcentrifuge tube provided in the kit Add 200 ul Elution Buffer and mix the buffer into the cellulose bed with a sterile pipette tip Centrifuge for 30 seconds but DO NOT decant the liquid The mRNA is now in the eluate Add a second 200 ul of Elution Buffer to the column mix into the cellulose and centrifuge again for 30 seconds Steps 2 through 4 elutes the mRNA into the microcentrifuge tube Remove the column from the tube The tube now contains 400 ul Do Not Discard This is your mRNA sample Precipitate the mRNA with 0 15 volume 60 ul of 2 M sodium acetate supplied and 2 5 volume 1 ml of 200 proof 100 ethanol Place on dry ice for 10 15 minutes Centrifuge in a microcentrifuge at maximum speed for 15 minutes at 4 C Carefully remove the supernatant and discard it without disturbing the pellet Wash the pellet with 70 ethanol Centrifuge again to collect any residual ethanol Remove the ethanol Centrifuge briefly and remove traces of ethanol Air dry the pellet for 5 10 minutes Resuspend the RNA pellet in 20 50 ul of Elution Buffer 10 mM Tris pH 7 5 Determin
9. brook J Fritsch E F Maniatis T 1989 Molecular Cloning A Laboratory Manual Cold Spring Harbor Laboratory 2nd edition For FastTrack Kit citations visit our citation database on www invitrogen com 1999 2006 Invitrogen Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use 14 e invitrogen
10. dbooks 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 Japanese Headquarters European Headquarters Invitrogen Corporation Invitrogen Japan Invitrogen Ltd 1600 Faraday Avenue LOOP X Bldg 6F Inchinnan Business Park Carlsbad CA 92008 USA 3 9 15 Kaigan 3 Fountain Drive Tel 1 760 603 7200 Minato ku Tokyo 108 0022 Paisley PA4 9RF UK Tel Toll Free 1 800 955 6288 Tel 81 3 5730 6509 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Fax 81 3 5730 6519 Tech Fax 44 0 141 814 6117 E mail tech_support invitrogen com E mail jpinfo invitrogen com E mail eurotech invitrogen com MSDS Requests Limited Warranty MSDSs Material Safety Data Sheets are available on our website at www invitrogen com msds Invitrogen is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 100 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 o
11. e the concentration of the mRNA see next page The mRNA may be used immediately or stored indefinitely at 80 C Basic mRNA Isolation Method Continued Determining RNA Yield FastTrack 2 0 Quick Reference Card To determine the concentration of the resuspended RNA dilute the sample 100 fold by adding 4 ul of sample to 396 wl of Elution Buffer Use Elution Buffer to blank the spectrophotometer at 260 nm Place the diluted sample into a 500 ul quartz cuvette and read the absorbance at 260 nm Determine the RNA concentration by using the following formula RNA A 0 04 ug ul D D is the dilution factor D 100 in the above example Determine the RNA yield by multiplying the concentration by the volume of the RNA Note that the A must be gt 0 05 to give an accurate RNA concentration 260 If you routinely use the FastTrack 2 0 mRNA Isolation Kit you may wish to use our laminated Quick Reference Card as a checklist Each step can be conveniently marked to keep track of centrifugations transfers and washes Use a laboratory marker pen to check off items and wipe off with ethanol to reuse Appendix Troubleshooting and Product Qualification Troubleshooting Product Qualification 12 The table below describes solutions to possible problems you might encounter If you need assistance at any time contact the Invitrogen Technical Support see next page Problem Reason Solution
12. f analysis The company will replace free of charge any product that does not meet those specifications This warranty limits Invitrogen 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 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 References Ausubel F M Brent R Kingston R E Moore D D Seidman J G Smith J A and Struhl K Ed 1990 Current Protocols in Molecular Biology Greene Publishing Associates and Wiley Interscience New York Sam
13. invitrogen FastTrack 2 0 Kit For isolation of mRNA Catalog nos K1593 02 K1593 03 Version K 4 December 2006 25 0099 ii Table of Contents INTO CIO ia aa rta cafeina 1 OVERVIEW ET en A Rn Lal nn ls a do dl en Ae ee ah 1 Materials A ds 3 Methods ici 5 Preparing Samples General Information ss 5 Preparing Samples Tissue Culture Cells sise 6 Preparing Samples Fresh and Frozen Tissue 7 Preparing Samples Plants and Total RNA eee 8 Basic MRNA Isolation Method dent a I alu 9 PAD PON A O 12 Troubleshooting and Product Qualification ss 12 Technical Support oeir nun ern en See etat 13 References is as 14 111 iv Overview Introduction Typical Yields Flow Chart of Process Introduction The FastTrack 2 0 mRNA Isolation Kit allows isolation of polyA RNA directly from cells 1 x 10 1x 10 tissue 0 4 1 0 g or total RNA 0 1 1 0 mg in 2 3 hours using minimal equipment water bath centrifuge microcentrifuge and homogenizer without the need for ultracentrifugation or guanidinium lysis The FastTrack 2 0 mRNA Isolation Kit yields the following amounts of mRNA e 40 70 ug per 1x10 cultured insect cells e 5 80 ug per gram of tissue e 10 20 ug per milligram of total RNA Yields of mRNA vary depending on the type of tissue and the stage of differentiation The following figure outlines the FastTrack process FastTrack 2 0 Kit 1 x 107 1 x 108 Tissue Culture
14. let the cells and continue with Step 3 or flash freeze the pellet in liquid nitrogen and store at 80 C Resuspend and lyse the cells by adding 15 ml FastTrack 2 0 Lysis Buffer page 5 to the cell pellet If the pellet was frozen and does not thaw quickly place the tube with the cells and buffer in a 45 C water bath for 1 2 minutes Vortex to completely resuspend the cells Pass the lysate 2 4 times through a sterile syringe fitted with a 18 21 gauge needle Proceed to the Basic mRNA Isolation Method Step 1 page 9 Preparing Samples Fresh and Frozen Tissue Preparing Tissue To achieve high yields of mRNA from plant insect or mammalian tissues use 0 5 for Storage 1 gram of tissue per isolation Perform all lysis and washing steps to ensure consistently high yields See page 8 for plant tissue preparation 1 Quickly excise the tissue cut into pieces of 1 2 cm with a sterile knife or razor blade and immediately freeze in liquid nitrogen 2 Store the tissue at 80 C in a polypropylene tube with a screw cap Frozen tissue is stable at 80 C for over 1 year Proceed to Preparing of Fresh or Frozen Tissue below Preparing Fresh or Thaw and homogenize the frozen tissue in the presence of lysis buffer to ensure Frozen Tissue immediate inactivation of any RNases that are released as the cells lyse Complete homogenization is critical for complete cell lysis and inactivation of RNases Be sure to clean and wash the h
15. omogenizer tip then autoclave and bake for 3 hours or overnight at 210 C 1 Place 1 g fresh or frozen tissue in a preweighed sterile 50 ml centrifuge tube and weigh Add 15 ml FastTrack 2 0 Lysis Buffer containing Proteinase K page 5 2 Quickly homogenize the tissue using a motor driven homogenizer such as Tissuemizer Teckmar or Omni Mixer Homogenizer Omni International Start at a low speed and slowly increase speed until a smooth homogenate with no visible particulate matter is obtained 15 30 seconds Keep foaming to a minimum by adjusting the speed 3 Proceed to the Basic mRNA Isolation Method Step 1 page 9 Alternative If a motor driven homogenizer is not available tissues may be homogenized using a Procedure mortar and pestle and a Dounce homogenizer Be sure to clean the equipment then autoclave and bake before use 1 Weigh out 1 g of fresh or frozen tissue and place into a mortar precooled with liquid nitrogen Cover the tissue with more liquid nitrogen and grind with a pestle until a fine powder is obtained Add more liquid nitrogen as needed to keep the tissue covered with liquid nitrogen while grinding 2 Transfer the suspension of liquid nitrogen and crushed tissue into a sterile 50 ml centrifuge tube containing 15 ml FastTrack 2 0 Lysis Buffer containing Proteinase K page 5 3 Transfer to a sterile Dounce homogenizer at room temperature Homogenize with 10 12 strokes Proceed to the Basic mRNA
16. rate a water bath to 45 C e Obtain dry ice for mRNA precipitation 1 Incubate the cell lysate produced in the sample prep procedures pages 6 8 at 45 C for 15 60 minutes If insoluble material persists centrifuge at 4 000 x g for 5 minutes at room temperature and transfer the supernatant to a new tube Incubation is important for complete digestion of proteins and ribonucleases A 60 minute incubation is recommended for tissues while cell material is effectively digested with a 15 minute incubation You may wish to optimize the time of incubation for your particular sample 2 Adjust the NaCl concentration of the lysate to 0 5 M final concentration by adding 950 ul of the 5 M NaCl stock solution to each 15 ml lysate containing 0 2 M NaCl Mix thoroughly by inversion 3 Shear any remaining DNA by passing the lysate 3 to 4 times through a sterile plastic syringe fitted with an 18 21 gauge needle This yields a cleaner mRNA preparation If solution is viscous see Troubleshooting on page 12 4 Remove a vial of oligo dT cellulose from the dessicator and add the contents of the vial to the lysate 5 Seal the tube and allow the oligo dT cellulose to swell for 2 minutes The oligo dT cellulose should disperse readily 6 Rock the tube gently at room temperature for 15 to 60 minutes Rocking or rotating increases the efficiency of mRNA binding to oligo dT cellulose 7 Pellet the oligo dT cellulose at 3 000 x g in a centrifuge for
17. terile 50 ml centrifuge tube 4 Heat to 65 C for 5 minutes then place immediately on ice for exactly 1 minute 5 Place the tube at room temperature and add 650 ul 5 M NaCl and mix by gentle inversion 6 Proceed to Step 4 of the Basic mRNA Isolation Method page 9 Basic mRNA Isolation Method Introduction Note Before Starting Isolating mRNA A typical mammalian cell contains about 107 g of RNA of which 1 5 is polyA RNA The remaining RNA is mostly rRNA 80 85 of total and low molecular weight RNAs such as tRNA 15 20 of total To separate the heterogeneous population of mRNA from the majority of the RNA found in the cell affinity binding to oligo dT cellulose is used This method exploits the major characteristic of mRNA polyadenylation to obtain intact pure mRNA The procedure described below is designed to remove DNA and proteins from your sample and allows selective binding of mRNA under high salt conditions to oligo dT cellulose The resin is then transferred to a microcentrifuge spin column and the rRNA is removed by washing the resin with a low salt buffer The mRNA is then eluted with a very low ionic strength buffer Intact pure mRNA is isolated due to the e Gentle lysis of cells e Efficient inactivation of RNases e Specificity of the oligo dT cellulose e Minimal handling of the mRNA sample The capacity of the oligo dT cellulose is 1 ug polyA RNA per mg of resin e Equilib
18. w to prepare the Lysis Buffer 1 Check the Stock Buffer for a white precipitate SDS If the Stock Buffer has a white precipitate heat the solution to 65 C until dissolved Cool the solution to room temperature prior to adding the Proteinase K 2 Add 300 pl Proteinase K to 15 ml Stock Buffer for each intended isolation Use immediately e If you are isolating polyA RNA from yeast cells note that the yield of polyA RNA is likely to be lower than the yields obtained with other eukaryotic cells Yeast cells generally yield lower amounts of polyA RNA than other eukaryotic cells because e Yeast mRNAs contain shorter polyA tails e Yeast cells are harder to lyse e To increase the yield of polyA RNA we recommend preparing total yeast RNA using PureLink Micro to Midi Total RNA Purification System Catalog no 12183 018 e When binding to oligo dT cellulose we recommend that you increase the incubation time of your RNA with oligo dT cellulose to at least 1 hour Increasing the incubation time of yeast RNA with the oligo dT cellulose may increase the binding efficiency Preparing Samples Tissue Culture Cells Preparing Tissue Cultured Cells We recommend using x 10 1 x 10 cells for each mRNA isolation 1 Wash the cells in 4 C phosphate buffered saline PBS solution available from Invitrogen visit www invitrogen com for details Transfer the cells to a 50 ml sterile centrifuge tube Pel
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