Connect with Epicentre on our blog epicentral
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Connect with Epicentre on our blog epicentral

blogspotcom Facebook facebookcomEpicentreBio and Twitter EpicentreBio Q EPILIT114 Rev A MasterPure RNA Puri64257cation Kit Cat No MCR85102 brPage 2br wwwepicentrecom MasterPure RNA Puri57375cation Kit 1 Introduction The MasterPu

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Connect with Epicentre on our blog epicentral




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Connect with Epicentre on our blog (epicentral.blogspot.com) , Facebook (facebook.com/EpicentreBio) , and Twitter (@EpicentreBio) Q    EPILIT114 Rev. A MasterPure™ RNA Purification Kit Cat. No. MCR85102
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www.epicentre.com MasterPure™ RNA Purication Kit 1. Introduction The MasterPure™ RNA Purication Kit pro vides all of the reagents necessary to recover RNA from a wide variety of biological sources. This kit uses a rapid desalting process to remove contaminating macromolecules, avoiding toxic organic solvents. The

puried RNA can be used subsequently in many applications including hybridization, RNase protection, and RT-PCR. We oer several products for PCR that incorporate the MasterAmp™ PCR Enhancement Technol ogy , which substantially improves product yield and decreases nonspecic product formation. 2. Product Specications Storage: Store the RNase-Free DNase I, Proteinase K, and RiboGuard™ RNase Inhibitor at ž$ Z  perature. Storage Buers: ' ̾* Q Q  Z  $ Q̾  $  $$ ; ProteinaseK is supplied in a  Z  $ Q   $  &"

 $$  ˇ    Quality Control: The MasterPure RNA Purica tion Kit is function-tested by purifying RNA from E.coli . RNA quality and yield are assayed by agarose gel electrophoresis, spectrophotometry, uorimetry, and use as a template for RT-PCR. 3. Kit Contents Desc. Concentration Quantity " ń Q  " puri cations. $ Z   $ Z    Z   $ Q   ' •  •    • – *  •  •  í   $  Q   & í  $  &" All MasterPure RNA Purication Kit com ponents are also available separately.
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techhelp@epicentre.com     MasterPure™ RNA Purication Kit 4. Related Products The following products are also available: MasterPure™ Complete DNA and RNA Purication Kits MasterPure™ DNA Purication Kit MasterPure™ Yeast DNA Purication Kit MasterPure™ Yeast RNA Purication Kit MasterPure™ Plant RNA Purication Kit "Q– " &Y "Q– 'Z $ "Q– $ Z MasterAmp™ PCR Optimization Kits MasterAmp Taq, Tth, T, and AmpliTherm™ DNA Polymerases FailSafe™ PCR System 5. General Considerations 1. Tissue Sources : We have used the kit

to isolate RNA from a variety of sources   plasma, saliva, corn and geranium leaf, yeast, E.coli , and lambda phage. Tissues other than those mentioned here are likely to be com patible with the kit with some optimization.  Isolation of RNA from Paran-Embedded Tissue: RNA isolated from preserved, paran-em bedded tissues is generally of poor quality. The degree of degradation of these samples limits anal ysis mainly to techniques involving amplication. To obtain RNA from embedded tissues that is amenable to RT-PCR, we recommend preserving   í ńY  

$ Q Qń Q ̾Q   Sample Size: You can purify nucleic acid from samples of various sizes by proportionally adjusting the amount of reagents to the amount of starting material. Q Q Z adjusted. 4. ProteinaseK Treatment: We recommend including the ProteinaseK treatment to increase the e ciency of lysis, though for some samples this treatment is Z   * Qń Z Z determine if ProteinaseK treatment is required. 5. Nuclease Treatment: The removal of DNA from RNA preparations with RNase- Free DNaseI is un necessary for many applications. This step may be

eliminated from the protocol depending upon the intended use of the RNA. If the removal of contaminating nucleic acid is necessary, we recommend performing these steps as outlined in the protocol. Note, however, for some samples, adjustments in nuclease concentration or time of incubation may improve the quality of the puried nucleic acid.
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www.epicentre.com MasterPure™ RNA Purication Kit 6. RNA Purication Protocols The following protocol is provided for the purication of RNA from several biological $  Z ó ̾" Q Q ̾ * Qń " " Q outlined

in PartC. Use appropriate techniques to minimize degradation by exogenous  " Qń Q Q̾ A. Lysis of Fluid or Tissue Samples Thoroughly mix the various Lysis Solutions to ensure uniform composition before dispensing. Fluid Samples  $ Q Q Z ž$       Z Q   ó Q   TandC Lysis Solution containing the ProteinaseK, and mix thoroughly. * ž$   Y Z   Q Q QQ ̾ Cell Samples  E.coli 1. Dilute 1    l of Tissue and Cell Lysis Solution for each sample.  Z      culture of E.coli

Q QQYZ  l of liquid. Y  Q Q "  l of Tissue and Cell Lysis Solution containing the ProteinaseK and mix thoroughly. * ž$   Y Z   Q Q QQ ̾ Tissue Samples  Q 1. Collect 1-5mg of tissue and either process immediately or freeze the samples at ž$      l of Tissue and Cell Lysis Solution for each sample.  microcentrifuge tube. "  l of Tissue and Cell Lysis Solution containing the ProteinaseK and mix thoroughly.
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techhelp@epicentre.com     MasterPure™ RNA Purication Kit *

ž$   Y Z   Q Q QQ ̾ Whole-Blood Samples $ Z   &" ˇ   l of whole blood into a micro centrifuge tube. Note: For processing larger volumes of whole blood, the volumes of MasterPure reagents can be scaled up. See Appendix A. "  l of Red Cell Lysis Solution. Invert three times to mix and then ick the bottom of the tube to suspend any remaining material. * Q Y óZ $ incubating at room temperature for an additional 5 minutes followed again by brief vortexing.  Z    Q QQYZ   Y suspend the pellet. Q  l of Tissue and Cell Lysis Solution by

pipetting the cells sev eral times.  Q Q QQ ̾ Formalin-Fixed, Paran-Embedded (FFPE) Tissues $ 1. Remove a section of tissue using a clean microtome blade; if possible, trim excess paran.      m thick paran sections into an appropriate tube. If using a larger amount of tissue, adjust the reagent volumes accordingly.    l of Tissue and Cell Lysis Solution for each sample, and mix. "  l of Tissue and Cell Lysis Solution containing the ProteinaseK to the sample and mix. * ž$   Z  Y Y  Q Q " QQ 
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www.epicentre.com MasterPure™ RNA Purication Kit B. Precipitation of Total Nucleic Acids Q "  $ Q  l of lysed sample and Y Z    Z ž$  Ŷ ̾Y microcentrifuge.  Q Q "  QQ Q *     Z ž$  microcentrifuge. $Z Q í QQ Q If removal of con taminating DNA from the RNA is required, proceed with PartC  Q ̾   pellet. Centrifuge briey if the pellet is dislodged. Remove all of the residual ethanol with a pipet. Q  & í C. Removal of Contaminating DNA from Total Nucleic Acid Preparations Q 1. Remove all

of the residual isopropanol with a pipet. Q  l of DNaseI solution for each sample by diluting 5 l of RNase-Free ̾* Q   í $QZ Q Q  l of DNaseI solution. * ž$   Note: Additional incubation (up to 30 minutes) may be necessary to remove all contaminating DNA. "   Z  Y  "  $ Q  Y   Q   Z ž$  Ŷ ̾Y microcentrifuge.  Q " discard the pellet. "  QQ Q *     Qń " Z ž$   11. Carefully pour o the isopropanol without dislodging the RNA pellet.   Q $ briey if the

pellet is dislodged. Remove all of the residual ethanol with a pipet. Q "   & í 14. Add 1 – * Q 
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techhelp@epicentre.com     MasterPure™ RNA Purication Kit 7. Additional Purication Protocols The following protocol is provided for the purication of RNA from plasma. If further Qń " " Q Q̾ QQQ Z Y ribonucleases. D. Lysis of Plasma Thoroughly mix the Tissue and Cell Lysis Solution to ensure uniform composition before dispensing. $ Q Q  l of plasma into a microcentrifuge tube.      l of Tissue and Cell Lysis

Solution for each sample. "  l of Tissue and Cell Lysis Solution containing the Proteinase K and mix thoroughly. * ž$   Y Z   QQ ̾&  E. Precipitation of Total Nucleic Acids Q Z 1. Place the samples on ice for 5 minutes. "  l of MPC Protein Precipitation Reagent and vortex mix vigorously for    Z ž$  Ŷ ̾Y microcentrifuge. 4. Transfer the supernatant to a clean microcentrifuge tube and discard the pellet. "  QQ Q *     " Z ž$   $Z Q í QQ " Q * " Q ̾+ Q̾  Q the remainder of PartE. 

 Q $ briey if the pellet is dislodged. Remove all of the residual ethanol with a pipet. Q "  & í
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www.epicentre.com MasterPure™ RNA Purication Kit The following protocol is provided for the purication of RNA from whole blood Z  * Qń " " Q ̾+ QQQ technique to minimize degradation by exogenous ribonucleases. F. Lysis of Whole Blood $ Z Thoroughly mix the Tissue and Cell Lysis Solution to ensure uniform composition before dispensing. $ Q  l of blood into a microcentrifuge tube.      l of Tissue and Cell Lysis Solution for

each sample. "  l of Tissue and Cell Lysis Solution containing the Proteinase K and mix thoroughly. * ž$   Y Z  5. Proceed with total nucleic acid precipitation in PartG. G. Precipitation of Total Nucleic Acids Z 1. Place the samples on ice for 5 minutes. "  $ Q Y Z  seconds.  Z ž$  Ŷ ̾Y microcentrifuge. 4. Transfer the supernatant to a clean microcentrifuge tube and discard the pellet. "  QQ Q *     Z ž$   $Z Q í QQ Q * " " QQ Q Q ̾+ Q̾  Otherwise, complete the remainder of PartG. 

 Q $ briey if the pellet is dislodged. Remove all of the residual ethanol with a pipet. Q  & í
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techhelp@epicentre.com     MasterPure™ RNA Purication Kit The following protocol is provided for the purication of RNA from the buy coat of  * Qń " " the protocol outlined in PartJ. Use appropriate techniques to minimize degradation by exogenous ribonucleases. H. Lysis of Buy Coat Thoroughly mix the various Lysis Solutions to ensure uniform composition before dispensing.   &"  Q Z  ̾Y  Z  l of

buy Q microcentrifuge tube. Transfer of some red blood cells is not detrimental to the Qń íZ  Y íZ Q  l of the sample to another microcentrifuge tube; process the two tubes in parallel. "  $ Z Y ó the bottom of the tubes to suspend any remaining material. * Q Y óZ $ incubating at room temperature for an additional 5 minutes, followed again by brief vortexing.  Z    Q QQYZ   Y suspend the pellets. Q  l of Tissue and Cell Lysis Solution by pipetting the cells sev eral times.  Q Q QQ ̾*  I. Precipitation of Total Nucleic Acids íZ "  l of MPC

Protein Precipitation Reagent and vortex vigorously for    Z ž$  Ŷ ̾Y microcentrifuge.  Q Q "  QQ Q *     Z ž$   $Z Q í QQ Q * " from RNA prepara tions is required, proceed with the protocol in PartJ. Otherwise, complete the remainder of PartI.   Q $ briey if the pellet is dislodged. Remove all of the residual ethanol with a pipet. Q  & í
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10 www.epicentre.com MasterPure™ RNA Purication Kit MasterPure™ RNA Purication Kit J. Removal of Contaminating DNA from RNA

Preparations 1. Remove all of the residual isopropanol with a pipet. Q  ̾* Q Z  l of RNase-Free ̾* Q   í $QZ Q Q  l of DNaseI solution. * ž$   "   Z  Y  "  $ Q  Y ̾ Q   Z ž$  Ŷ ̾Y microcentrifuge.  Q " discard the pellet. "  QQ Q *     Qń " Z ž$   11. Carefully pour o the isopropanol without dislodging the RNA pellet.   Q $ briey if the pellet is dislodged. Remove all of the residual ethanol with a pipet. Q "   & í 14. Add 1 * Q  8.

Troubleshooting RNA Purications Little or no RNA after resuspension in TE buer   Increase the amount of tissue or biological uid. Use the recommended amount of starting material or use the recommended ratio of tissue:lysis buer as indicated in the protocol. Increase the amount of tissue, particularly if purifying RNA from a biological source other than those listed in the protocols.   Increase the eciency of cell lysis. Either increase the amount of ProteinaseK used during lysis or increase the time of incubation. In addition, vortex during

ProteinaseK treatment to facilitate lysis. If these adjustments fail, homogenize the tissue to more fully disrupt the cell membrane or wall.   Decrease the amount of TE buer. & í Q QQ RNA.   Avoid contamination by exogenous or endogenous nucleases. Ensure that tissue or biological uids were properly collected and stored. Use sterile technique. Add & í Q   Ensure that RNA remains following isopropanol precipitation. Make certain that the RNA pellet adheres to the microcentrifuge tube during washing of the pellet  
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techhelp@epicentre.com 

   11 MasterPure™ RNA Purication Kit 260 /A 280 ratio is too low   Decrease the amount of starting material. The RNA is contaminated with protein. ó Z "  l with Tissue and Cell Lysis Solution, and re peat the purication protocol. Loose protein pellet   Cool sample before protein precipitation. Cool the sample thoroughly on ice before adding the MPC Protein Precipitation Reagent. If the pellet remains loose, centrifuge again. Carefully decant to minimize transfer of precipitated protein. Note that a small degree of transfer is generally not detri mental. 9.

Reference 1. Miller, S.A. et al.  Nucl. Acids Res. 16  Covered by U.S. Patent No. 6,270,962, European Patent No. 0742838, German Patent No. DE4411588C1, and other issued or pending applications in the U.S. and other countries that are either assigned or exclusively licensed to Epicentre. AmpliTherm, BuccalAmp, FailSafe, MasterAmp, MasterPure, and RiboGuard are trademarks of Epicentre, Madison, Wisconsin. Triton is a registered trademark of Rohm & Haas, Philadelphia, Pennsylvania. Vacutainer is a registered trademark of Becton Dickinson Corp., Rutherford, New Jersey. 

epicentral.blogspot.com
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techhelp@epicentre.com     12 MasterPure™ RNA Purication Kit Whole Blood Tube Size Red Cell Lysis Solution Tissue and Cell Lysis Solution MPC Isopropanol  1.5 ml      15 ml    1.5 ml  15 ml     1 ml 15 ml  1.5 ml   15 ml     15 ml  4.5 ml   4 ml      5 ml  15 ml  4.4 ml      15 ml           9 ml         15 ml         DNase I DNase I Buer 2X T & C MPC

Isopropanol Ribo-Guard 5 ml     1 ml 15 ml    1.5 ml       1 ml 1 ml 1 ml      5 ml  4 ml 4 ml 4 ml   5 ml 5 ml 5 ml   15 ml       9 ml 9 ml 9 ml               10. Appendix A MasterPure™ RNA Purication Kit $  $ Purication of RNA from Whole Blood: MasterPure Reagent Volume Chart