TRIzol Max Bacterial RNA Isolation Kit

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TRIzol® Max™ Bacterial RNA Isolation Kit Catalog nos. 16122-012, 16096-020, 16096-040 Description Types of Products This manual is supplied with the following products. Twenty milliliters of Max Bacterial Enhancement Reagent is sufficient to perform 100 isolations. Product Max Bacterial Enhancement Reagent TRIzol Max Bacterial RNA Isolation Kit ® ™ Contents 20 ml 20 ml Max Bacterial Enhancement Reagent 100 ml TRIzol® Reagent 2 x 20 ml Max Bacterial Enhancement Reagent 200 ml TRIzol® Reagent C
   TRIzol  ®    Max ™ Bacterial RNA Isolation Kit Catalog nos. 16122-012, 16096-020, 16096-040 Description Types of Products This manual is supplied with the following products. Twenty milliliters of Max BacterialEnhancement Reagent is sufficient to perform 100 isolations. Product Contents Catalog no. Max Bacterial Enhancement Reagent 20 ml 16122-012TRIzol ® Max ™ Bacterial RNA Isolation Kit 20 ml Max BacterialEnhancement Reagent100 ml TRIzol ® Reagent16096-020TRIzol ® Max ™ Bacterial RNA Isolation Kit 2 x 20 ml Max BacterialEnhancement Reagent200 ml TRIzol ® Reagent16096-040 Storage The Max Bacterial Enhancement Reagent and TRIzol ® Reagent are shipped at room temperature.Store Max Bacterial Enhancement Reagent at room temperature. Do not store at 4 ° C or -20 ° C. If aprecipitate is formed, heat at 65 ° C until precipitate is dissolved.Store TRIzol ® Reagent at 4 ° C. Description The Max Bacterial Enhancement Reagent is designed for use with TRIzol ® Reagent to improve theisolation of intact total RNA up to 3-fold from Gram-positive and Gram-negative bacteria. The MaxBacterial Enhancement Reagent is a ready-to-use solution composed of chelating agents, detergent, anda buffer, and is used as an efficient pre-treatment buffer for bacterial cells prior to RNA isolation withTRIzol ® Reagent. The use of Max Bacterial Enhancement Reagent with TRIzol ® inactivates endogenousRNases and promotes protein denaturation improving the RNA quality and integrity.Bacterial cells are pre-treated with Max Bacterial Enhancement Reagent and incubated at hightemperature. TRIzol ® Reagent is then added to dissolve the cell components and maintain RNAintegrity. Addition of chloroform followed by centrifugation separates the lysate into an aqueousphase containing RNA and an organic phase. RNA is recovered from the aqueous phase byprecipitation with isopropanol. The final RNA pellet is dissolved inRNase free/DEPC-treated water. Advantages Using Max Bacterial Enhancement Reagent with TRIzol ® Reagent to isolate total RNA from bacteriaoffers the following advantages: ã Higher yields due to improved lysis of bacterial cells and minimal RNA degradation ã Designed to improve total RNA isolation from Gram-positive and Gram-negative bacteria ã Minimal genomic DNA contamination of the purified RNA sample ã Eliminates the need for time consuming mechanical and/or enzymatic cell lysis steps ã Reliable performance of the high-quality purified total RNA in downstream applicationsPart No. 25-0669 Rev. Date: 07/25/03  Page 2 Using Max Bacteria l Enhancement Reagent When working with TRIzol ® Reagent, use a laboratory coat, safety glasses, and gloves. Avoid contactwith skin or clothing. Use in a chemical fume hood and avoid breathing vapor. MaterialsNeeded You will need the following items: ã TRIzol ® Reagent (supplied with catalog nos. 16096-020 and 16096-040) ã Bacterial cells ã Chloroform, chilled ã Isopropanol, chilled ã 75% Ethanol ã RNase-free water (page 3) ã Heating block set at 95 ° C ã Microcentrifuge and sterile microcentrifuge tubes GeneralHandling of RNA Observe the following guidelines to prevent RNase contamination: ã Use disposable, individually wrapped, sterile plasticware ã Use only sterile, new pipette tips and microcentrifuge tubes ã Wear latex gloves while handling reagents and RNA samples to prevent RNase contaminationfrom the surface of the skin ã Always use proper microbiological aseptic techniques when working with RNA ã Use RNase AWAY ® Reagent (page 3) to remove RNase contamination from surfaces Procedure 1. Inoculate bacteria into a suitable medium (add appropriate antibiotic, if needed).2. Incubate the culture with shaking at the appropriate temperature for the desired time to obtain logphase cells.3. Transfer 1.5 ml of bacterial culture (up to 1 x 10 8 cells) to a pre-chilled microcentrifuge tube.4. Centrifuge the tube at 6000 x g for 5 minutes at 4 ° C in a microcentrifuge.5. During centrifugation, preheat 200 µ l Max Bacterial Enhancement Reagent to 95 ° C.6. After centrifugation, decant the supernatant and resuspend the cell pellet in preheated 200 µ l MaxBacterial Enhancement Reagent from the previous step. Mix well by pipetting up and down.7. Incubate at 95 ° C for 4 minutes.8. Add 1 ml TRIzol ® Reagent to the lysate and mix well.9. Incubate at room temperature for 5 minutes. Proceed to Phase Separation  , below. PhaseSeparation 1. Add 0.2 ml cold chloroform and mix by shaking the tube vigorously by hand for 15 seconds.2. Incubate at room temperature for 2-3 minutes.3. Centrifuge the samples 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 aqueous phase containing RNA. The volume of the aqueous phase is ~400 µ l.   Proceed to Precipitating RNA  , next page.  Note: Isolation of DNA and proteins from the interphase and phenol phase after RNA isolation has not been tested and is not recommended with the Max Bacterial Enhancement Reagent.  Page 3 Using   Max Bacteria l Enhancement Reagent   PrecipitatingRNA 1. Transfer ~400 µ l of the colorless, upper phase containing RNA to a fresh tube.2. Add 0.5 ml cold isopropanol to the aqueous phase to precipitate RNA. Mix by inverting the tube.3. Incubate at room temperature for 10 minutes.4. Centrifuge at 15,000 x g for 10 minutes at 4 ° C.5. Remove the supernatant carefully without disturbing the RNA pellet (a gel-like pellet formed atthe side and bottom of the tube).6. Resuspend the pellet in 1 ml 75% ethanol. Mix well by vortexing.7. Centrifuge at 7,500 x g for 5 minutes at 4 ° C.8. Air-dry the RNA pellet. Do not dry the RNA pellet by centrifugation under vacuum.9. Resuspend the RNA pellet in 50 µ l RNase-free water by pipetting up and down and incubating for10 minutes at 60 ° C, if needed. EstimatingRNAQuantity Determine the purified total RNA quantity as described below.1. Dilute an aliquot of the total RNA sample in 10 mM Tris-HCl, pH 7.0. Mix well. Transfer to a 1-cmpath length cuvette.2. Determine the absorbance of the solution at 260 nm using a spectrophotometer blanked against 10mM Tris-HCl, pH 7.0.3. Calculate the amount of total RNA using the following formula:Total RNA ( µ g) = A 260 x 40 µ g/(1 A 260 x 1 ml) x dilution factor x total sample volume (ml) Expected Yield The yield of total RNA isolated from 1.5 ml bacterial cells (~1x 10 8 cells) using the Max BacterialEnhancement Reagent with TRIzol ® Reagent is >20 µ g for E. coli (Gram-negative bacteria) and ~3 µ g for Lactococcus lactis (Gram-positive bacteria).Agarose gel electrophoresis of the purified RNA shows distinct 16S and 23S ribosomal bands. Trouble-shooting Review the information below to troubleshoot your experiments with the Max Bacteria EnhancementReagent. Problem Cause Solution Incomplete lysisIncubate the sample at 95 ° C for 5 minutes afteradding Max Reagent to facilitate cell lysis.Incorrect phase transferred The RNA is in the colorless, aqueous phase.Use this phase for precipitating RNA.Low RNAyieldIncomplete dissolution of the final RNA pelletBe sure to completely dissolve the final RNApellet. If needed, heat at 60 ° C for 10 minutes.RNAdegradedRNase contamination Follow the guidelines on page 2 to preventRNase contamination. AccessoryProduct The following products are available from Invitrogen. For more details on these products, visit ourWeb site or contact Technical Service. Product Quantity Catalog no. TRIzol ® Reagent 100 ml 15596-026200 ml 15596-018RNase AWAY ® 250 ml 10328-011UltraPure ™ DNase/RNase-Free Distilled Water 500 ml 10977-015UltraPure ™ DEPC-treated Water 1 L 750023   Page 4 Product Qualification and Purchaser Notification ProductQualification The Max Bacterial Enhancement Reagent and TRIzol ® Reagent are functionally qualified by isolatingtotal RNA from ~1 x 10 8   E. coli cells as described in this manual and must produce the following results: ã A 260/280 between 1.85 and 2.15 ã Intact RNA as determined by visual inspection on an agarose gel ã Yield must be >20 µ gIn addition, Max Bacterial Enhancement Reagent and TRIzol ® Reagent are guaranteed sterile and freeof ribonuclease contamination. Limited UseLabelLicense No.164: LysisEnhancement Buffer   The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of theproduct and components of the product in research conducted by the buyer (whether the buyer is an academic orfor-profit entity). The buyer cannot sell or otherwise transfer (a) this product (b) its components or (c) materials madeusing this product or its components to a third party or otherwise use this product or its components or materialsmade using this product or its components for Commercial Purposes. The buyer may transfer information ormaterials made through the use of this product to a scientific collaborator, provided that such transfer is not for anyCommercial Purpose, and that such collaborator agrees in writing (a) not to transfer such materials to any thirdparty, and (b) to use such transferred materials and/or information solely for research and not for CommercialPurposes. Commercial Purposes means any activity by a party for consideration and may include, but is not limitedto: (1) use of the product or its components in manufacturing; (2) use of the product or its components to provide aservice, information, or data; (3) use of the product or its components for therapeutic, diagnostic or prophylacticpurposes; or (4) resale of the product or its components, whether or not such product or its components are resoldfor use in research. Invitrogen Corporation will not assert a claim against the buyer of infringement of patents owned by Invitrogen Corporation and claiming this product based upon the manufacture, use or sale of a therapeutic,clinical diagnostic, vaccine or prophylactic product developed in research by the buyer in which this product or itscomponents was employed, provided that neither this product nor any of its components was used in themanufacture of such product. If the purchaser is not willing to accept the limitations of this limited use statement,Invitrogen is willing to accept return of the product with a full refund. For information on purchasing a license tothis product for purposes other than research, contact Licensing Department, Invitrogen Corporation, 1600 FaradayAvenue, Carlsbad, California 92008. Phone (760) 603-7200.Fax (760) 602-6500. LimitedWarranty Invitrogen is committed to providing our customers with high-quality goods and services. Our goal is to ensure thatevery customer is 100% satisfied with our products and our service. If you should have any questions or concernsabout an Invitrogen product or service, contact our Technical Service 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 Invitrogen Corporation’sliability only to the cost of the product. No warranty is granted for products beyond their listed expiration date. Nowarranty is applicable unless all product components are stored in accordance with instructions. Invitrogen reservesthe right to select the method(s) used to analyze a product unless Invitrogen agrees to a specified method in writingprior to acceptance of the order. Invitrogen makes every effort to ensure the accuracy of its publications, but realizesthat the occasional typographical or other error is inevitable. Therefore Invitrogen makes no warranty of any kindregarding the contents of any publications or documentation. If you discover an error in any of our publications,please report it to our Technical Service Representatives. Invitrogen assumes no responsibility or liability for anyspecial, incidental, indirect or consequential loss or damage whatsoever. The above limited warranty is sole andexclusive. No other warranty is made, whether expressed or implied, including any warranty of merchantabilityor fitness for a particular purpose.    2003 Invitrogen Corporation. All rights reserved. TRIzol ® is a registered trademark of Molecular Research Center, Inc. RNase AWAY ® is a registered trademark of Molecular Bio-Products, Inc.  
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