Real-Time PCR - Gene-Quantification.info
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1. Invitrogen life technologies Instruction Manual LUX Fluorogenic Primers For real time PCR and RT PCR Version B 091802 25 0546 ii Table of Contents ig A A A EN 1 Designing and Ordering PAM inci teta 3 Storing and Reconstituting PIIMOlSsisderseceddericcedderSecndderlecedderbecedderdacedderbecedderbacndderSecn derss 5 Real Time POR ii ER ee AEE Renee Re 6 Multiplex Real Hime POR T 8 Two Step Real Time TP Rus 9 One Step Real Time RT PCR liada 11 Troubleshooting 00 ito 14 Accessory Sele 1 6 C 15 Purchaser NOMINA Ras 16 Rena eT ale 17 References nde tienes edie AA Eee 19 iii iv Introduction Overview LUX Primer Reaction Labeling LUX Light Upon Extension primers are an easy to use highly sensitive and efficient method for performing real time quantitative PCR and RT PCR LUX primers combine high specificity and multiplexing capability with simple design and streamlined protocols LUX primers are compatible with melting curve analysis of real time PCR products allowing the differentiation of amplicons and primer dimer artifacts by their melting temperatures LUX primers require no special probes or quenchers and can be ordered direct from Invitrogen TM Each primer pair in the LUX system includes a fluorogenic primer with a fluorophore attached to its 3 end as well as a corresponding unlabeled primer The fluorogenic primer has a short sequence tail2. We recommend using a hot start DNA polymerase preferably one that has been optimized for real time PCR Platinum Quantitative PCR SuperMix UDG available from Invitrogen Catalog no 11730 017 is a 2X concentrated ready to use reaction mixture containing all components except primers and template It uses Platinum Tag DNA polymerase and has been specifically formulated to provide optimal performance in real time PCR systems See the sample protocol below The following optimized reaction uses Platinum Quantitative PCR SuperMix UDG with ROX reference reagent Note Before proceeding see the real time PCR guidelines on the previous page For multiplex reactions see the guidelines on the following page 1 To reduce pipetting errors prepare a Master Mix of all the reaction ingredients except template The following table provides Master Mix volumes for one reaction and 50 reactions scale up or down as needed Component Stock conc Vol 1 rxn Vol 50 rxns Rxn conc Platinum Quantitative PCR SuperMix UDG 2X 25 ul 1250 ul 1X ROX reference dye 50X 1 ul 50 ul 1X Sterile distilled H O 12 ul 600 ul Reverse primer 10 uM Tul 50 ul 200 nM Forward primer 10 uM _1ul 50 ul 200 nM Total volume 40 ul 2000 ul Supplied at 2X concentration 60 U ml Platinum Taq DNA polymerase 40 mM Tris HCl pH 8 4 100 mM KCI 6 mM MgCl 400 uM dGTP 400 uM dATP 400 uM dCTP 800 uM dUTP 40 U ml UDG and stabiliz
3. Technical Resources and select MSDS Requests Follow instructions on the page and fill out all the required fields To request additional MSDSs click the Add Another button All requests will be faxed unless another method is selected SL E 9 MD When you are finished entering information click the Submit button Your MSDS will be sent within 24 hours Continued on next page 17 Technical Service Continued Limited Warranty 18 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 please contact our Technical Service Representatives Invitrogen warrants that all of its products will perform according to the 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 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 Inv
4. Products The following products are available for use with LUX primers in real time PCR and RT PCR protocols Platinum Quantitative PCR SuperMix UDG 100 rxns 11730 017 500 rxns 1 ans Platinum Quantitative RT PCR ThermoScript 100 rxns 015 Platinum Tag DNA Polymerase 100 rxns 10966 018 250 rxns 10966 026 500 rxns 10966 034 5 000 rxns 10966 083 Micro to Midi Total RNA Purification Micro to Midi Total RNA Purification System 50rxns rxns 12183 018 018 A Reagent 100 E S 026 200 ml 15596 018 Micro FastTrack 2 0 mRNA Isolation Kit K1520 02 ROX Reference Dye 500 pl 12223 023 DNase I DNase I Amplification Grade 1 U W Grade 1 U l J100 U 10868 015 015 i E Recombinant Ribonuclease Inhibitor EAT 000 U m 019 40 U ul 10 mM dNTP Mix 100 ul 18427 013 DEPC treated water 4x1 25 ml 10813 012 15 Purchaser Notification Limited Use Label License No 114 LUX Fluorogenic Primer Limited Use Label License No 4 Products for PCR which do not include any rights to perform PCR 16 The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or o
5. PCR programs with annealing temperatures from 55 to 64 C are appropriate After you submit your sequence LUX Designer will first generate one or more designs for the labeled primer The labeled primer can be either the forward or the reverse primer note the recommendation for one step RT PCR on the following page After you select a design for the labeled primer you will be prompted to select a design for the corresponding unlabeled primer Continued on next page Designing and Ordering Primers Continued a L N NO Y Sy CA BA u o G Z Selecting Labels Placing the Order Product Qualification In one step RT PCR see the sample reaction on page 11 the reverse primer drives the reverse transcription reaction We have found that labeling the reverse primer with the LUX fluorophore can inhibit this RT step therefore we strongly recommend that you select the forward primer as the labeled primer You should choose a forward primer as the labeled primer for one step RT PCR regardless of its penalty score If no forward primer can be generated for the sequence you may select the reverse primer as the labeled primer noting that the efficiency of the reaction may be compromised After you have selected a primer set labeled and unlabeled for a particular sequence you can specify the particular label and synthesis scale When selecting labels in a multiplex reaction we recommend using the FAM label fo
6. See the sample reaction on page 7 The optimal concentration of dATP dCTP dGTP and dTTP is 200 uM each If dUTP is used in place of dTTP its optimal concentration is 400 uM LUX primers are compatible with a wide variety of real time PCR instruments with various detection capabilities including but not limited to the ABI PRISM 7700 7000 7900 and GeneAmp 5700 the Bio Rad iCycler the Stratagene Mx4000 and the Cepheid Smart Cycler At a minimum the instrument used to perform real time PCR with LUX primers must be able to e Detect fluorescence at each PCR cycle e Excite FAM and JOE labeled LUX primers near their excitation wavelength maximums of 490 nm and 520 nm respectively e Detect the emission of FAM and JOE labeled LUX primers near their emission maximums of 520 nm and 550 nm respectively Please refer to the specific instrument s user manual for operating instructions Please follow the manufacturer s instructions for configuring your real time PCR instrument for use with LUX primers Note the following settings e Because LUX primers do not contain a quencher the quencher setting should be set to no quencher e We recommend the use of ROX reference dye Catalog no 12223 023 for normalization of well to well variation Adjust your instrument reference setting accordingly Continued on next page Real Time PCR Continued Enzyme Specifications Real Time PCR Protocol
7. Component Volume Platinum Quantitative PCR SuperMix UDG 2X 25 ul ROX reference dye 50X Tul Sterile distilled H O 10 ul Template 10 ul Forward primer 1 FAM label 10 uM Tul Reverse primer 1 10 uM 1ul Forward primer 2 JOE label 10 uM Tul Reverse primer 2 10 uM _1ul Total volume 50 ul Reduce the volume of water to compensate for the additional primer volume All other reaction volumes remain the same Follow the thermal cycling guidelines provided in the sample reaction on the previous page If you have difficulty performing the multiplex reaction using these guidelines see the optimization hints below If you notice a decline in real time PCR efficiency in your multiplex real time PCR you can optimize the reaction by performing the steps listed below Note We recommend that you perform one optimization step and then repeat the reaction to test for efficiency before moving on to the next step 1 Reduce the primer concentration of the gene with the highest abundance typically the housekeeping gene to 1 4 the primer concentration of the other gene For example in a standard 50 ul reaction you would add the primers for the less abundant gene at 1 ul each and add the primers for the more abundant gene at 0 25 ul each Increase the MgCl in the reaction from 3 mM to 5 mM Increase the dNTP concentrations in the reaction to 400 uM each Double the amount of polymerase enzyme to 0 06 U per ul of reaction volume I
8. of 4 6 nucleotides on the 5 end that is complementary to the 3 end of the primer The resulting hairpin secondary structure provides optimal quenching of the fluorophore see the figure below When the primer is incorporated into the double stranded PCR product the fluorophore is dequenched and the signal increases by up to 10 fold Relative fluorescence 0 1 Hairpin primer eee re 0 4 Single stranded primer Extended primer double stranded DNA Each fluorogenic LUX primer is labeled with one of two reporter dyes FAM 6 carboxy fluorescein or JOE 6 carboxy 4 5 dichloro 2 7 dimethoxy fluorescein Additional reporter dyes will be available in the future A multiplex real time reaction contains different primer sets each with a different label to detect different genes in the same sample Continued on next page Introduction Continued Applications Instrument Compatibility LUX primers can be used in real time PCR and RT PCR to quantify 100 or fewer copies of a target gene in as little as 1 pg of template DNA or RNA LUX primers have a broad dynamic range of 7 8 orders Multiplex applications use separate FAM and JOE labeled primer sets to detect two different genes in the same sample Typically the FAM labeled primer is used to detect the gene of interest and the JOE labeled primer is used to detect a housekeeping gene used as an internal control LUX primers are compati
9. review them select a design select the fluorophore labels and place your order When you submit a target sequence containing your gene of interest keep in mind the following design criteria e The optimal amplicon length for real time PCR ranges from 80 to 200 bases You can specify a minimum optimal and maximum amplicon length when you submit the sequence e The target sequence should be at least 10 bases longer than the minimum amplicon size you select The longer the sequence the more likely that an optimal primer design can be developed e The sequence must contain only standard IUPAC International Union of Pure and Applied Chemistry letter abbreviations e When you first submit a sequence the Disable Penalty Checking checkbox should not be checked the resulting penalty scores provide an important measure of primer suitability Penalty scores in the range of 0 0 4 0 are acceptable If no primers with a penalty score of 4 0 or lower are generated you may choose to disable Penalty Checking and redesign the primers Note that if you select a primer with a higher penalty score the efficiency of the reaction may be less than optimal See the LUX Designer Help for guidance in optimizing your design parameters and or sequence s When you select the design parameters the default melting temperature range is 60 68 C Do not change this default unless the design engine finds no primers in this range For primers in this range
10. MgCl 4ul 10 mM dNTP 1 ul 0 1M DTT 2 ul RNaseOUT 40 U ul 1 ul SuperScript II RT 50 U ul 1 ul DEPC treated ddH O to 20 ul Incubate tube at 25 C for 10 min Incubate tube at 42 C for 30 50 min Terminate the reaction at 70 C at 15 min and then chill on ice SL RQ ID Optional RNaseH treatment Add 1 ul of RNaseH and incubate at 37 C for 20 min Store the reaction at 20 C Use 2 8 ul of cDNA for real time PCR using Platinum Quantitative PCR SuperMix UDG as described on pages 6 7 10 One Step Real Time RT PCR Introduction Eco gt Z z S Nos l Note Template Specifications Enzyme Specifications Primer Concentration Magnesium Concentration dNTP Concentration This section provides information and a generic protocol for performing one step real time RT PCR using LUX primers Note that one step RT PCR is a complex reaction that may require additional optimization and evaluation of several primer pairs to obtain optimum sensitivity and specificity In one step RT PCR the reverse primer drives the reverse transcription reaction We have found that labeling the reverse primer with the LUX fluorophore can inhibit this RT step therefore we strongly recommend that you label the forward primer with the FAM or JOE fluorophore See pages 3 4 for guidance on primer design LUX primers have not been tested in multiplex one step real time RT PCR The target te
11. als in Adobe Acrobat PDF format e Explore our catalog with full color graphics e Obtain citations for Invitrogen products e Request catalog and product literature Once connected to the Internet launch your Web browser Internet Explorer 5 0 or newer or Netscape 4 0 or newer then enter the following location or URL http www invitrogen com and the program will connect directly Click on underlined text or outlined graphics to explore Don t forget to put a bookmark at our site for easy reference Contact Us For more information or technical assistance please 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 K K Invitrogen Ltd 1600 Faraday Avenue Nihonbashi Hama Cho Park 3 Fountain Drive Carlsbad CA 92008 Bldg 4F Inchinnan Business Park USA 2 35 4 Hama Cho Nihonbashi Paisley PA4 9RF UK Tel 1 760 603 7200 Tel 81 3 3663 7972 Tel 44 0 141 814 6100 Tel Toll Free 1 800 955 6288 Fax 81 3 3663 8242 Tel Toll Free in UK Fax 1 760 602 6500 E mail jpinfo invitrogen com 0800 5345 5345 E mail Fax 44 0 141 814 6287 tech service invitrogen com E mail eurotech invitrogen com MSDS Requests To request an MSDS please visit our Web site www invitrogen com and follow the instructions below 1 On the home page go to the left hand column under
12. ble with a wide variety of real time PCR instruments including but not limited to the ABI PRISM 7700 7000 7900 and GeneAmp 5700 the Bio Rad iCycler the Stratagene Mx4000 and the Cepheid Smart Cycler ABI PRISM is a registered trademark of Applera Corporation GeneAmp is a registeredtrademark of Roche Molecular Systems Inc iCycler Mx4000 and Smart Cycler are trademarks of their respective companies Designing and Ordering Primers LUX Designer Web based Design Software Submitting a Target Sequence Selecting a Primer Design TM To design and order LUX primers visit the Invitrogen LUX Web site at www invitrogen com LUX and follow the link to the LUX Designer Web based design software Follow the step by step instructions in the software to submit target sequences containing your genes of interest and generate primer designs At any point in the process click on the LUX Designer Help button for more detailed instructions and assistance LUX Designer will automatically generate one or more primer designs based on each sequence you submit and the selected design parameters The design software includes algorithms to minimize primer self complementarity and interactions between primers It also assigns rankings to the generated designs based on primer melting temperature hairpin structure self annealing properties etc to aid in selection When the designs have been generated you can
13. ers or use DNase RNase Free Distilled Water Cat No 10977 015 2 Program the real time PCR instrument with one of the following thermal cycling protocols 3 Step Cycling recommended 2 Step Cycling optional 50 C 2 min hold 50 C 2 min hold 95 C 2 min hold 95 C 2 min hold 45 cycles of 45 cycles of 95 C 15 s 95 C 15s 55 C 30 s 60 65 C 30 45 s 72 C 30 s 3 Add 40 ul of the Master Mix to an optical PCR tube or each well of a 96 well PCR plate 4 Add 10 ul of template diluted in TE or sterile dH O to the tube or each well of the 96 well PCR plate Cap or seal the tube plate 5 Gently mix and make sure that all components are at the bottom of the tube plate wells Centrifuge briefly if needed 6 Place reaction in the real time PCR instrument and run the program Collect and analyze results Multiplex Real Time PCR Multiplex Real Time PCR Optimizing Multiplex Conditions In multiplex real time PCR different sets of primers with different labels are used to amplify separate genes on the template DNA LUX primers have been tested in multiplex reactions using FAM to label the gene of interest and JOE to label a housekeeping gene used as an internal control to normalize between different reactions In a standard multiplex reaction you can include the additional primers at the same volumes and concentration as the primers in a singleplex reaction as shown in the example mixture below
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15. f this limited use statement Invitrogen is willing to accept return of the products with a full refund For information on purchasing a license to this product for purposes other than research contact Licensing Department 1600 Faraday Avenue Carlsbad California 92008 Phone 760 603 7200 Fax 760 602 6500 This product is optimized for use in the Polymerase Chain Reaction PCR covered by patents owned by Roche Molecular Systems Inc and F Hoffmann La Roche Ltd Roche No license under these patents to use the PCR process is conveyed expressly or by implication to the purchaser by the purchase of this product A license to use the PCR process for certain research and development activities accompanies the purchase of certain reagents from licensed suppliers such as Invitrogen when used in conjunction with an Authorized Thermal Cycler or is available from Applied Biosystems Further information on purchasing licenses to practice the PCR process may be obtained by contacting the Director of Licensing at Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 or at Roche Molecular Systems Inc 1145 Atlantic Avenue Alameda California 94501 Technical Service World Wide Web Visit the Invitrogen Web Resource using your World Wide Web browser At the site you can e Get the scoop on our hot new products and special product offers e View and download vector maps and sequences e Download manu
16. f you are using Platinum Quantitative PCR SuperMix UDG add Platinum Taq DNA polymerase stand alone enzyme Catalog no 10966 018 to double the amount of enzyme Two Step Real Time RT PCR Introduction Template Specifications Enzyme Specifications Removing Genomic DNA from RNA Samples For real time RT PCR applications we recommend a two step protocol so that the RT and PCR modules can be optimized separately for maximum efficiency and specificity This section provides an optimized protocol for performing reverse transcription as part of a two step real time RT PCR protocol You can use the resulting cDNA in the real time PCR reaction on pages 6 7 The target template for real time RT PCR is RNA usually total cellular RNA or mRNA The amount of RNA typically varies from 1 pg to 100 ng of template per assay The purity and integrity of the RNA have a direct impact on results RNase and genomic DNA contamination are the most common problems and purification methods should include RNase inhibitors and DNase digestion to minimize these We recommend using the Micro to Midi Total RNA Purification System Catalog no 12183 018 or TRIzol reagent Catalog no 15596 026 to isolate total RNA High quality total RNA can be purified from as little as 100 cells up to 10 cells or 200 mg of tissue To isolate mRNA we recommend using the FastTrack 2 0 mRNA Isolation Kit Catalog no K1593 02 We recommend usi
17. itrogen 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 Service 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 Ailenberg M and Silverman M 2000 Controlled hot start and improved specificity in carrying out PCR utilizing touch up and loop incorporated primers TULIPS BioTechniques 29 1018 1024 Bustin S A 2000 Absolute quantification of mRNA using real time reverse transcription polymerase chain reaction assays J Mol Endocrinol 25 169 193 Cardullo R A Agrawal S Flores C Zamecnik P C and Wolf D E 1988 Detection of nucleic acid hybridization by nonradiative fluorescence resonance energy transfer Proc Natl Acad Sci USA 85 8790 8794 Crockett A O and Wittwer C T 2001 Fluorescein labeled oligonucleotides for real time pcr using the inherent quenching of deoxyguanosine nucleotides Anal Biochem 290 89 97 Hig
18. l to well variation Adjust your instrument reference setting accordingly e Program the instrument to perform cDNA synthesis immediately followed by PCR amplification We recommend that you decrease the genomic DNA content in the RNA sample by performing a digest with DNase I Amplification Grade Catalog no 10868 015 as described below The DNase I digest is designed for up to 1 ug of RNA for larger amounts of RNA increase volumes accordingly Combine the following in a tube on ice Component Conc Volume RNA template x pl DNase reaction buffer 10X 1 ul DNase I Amplification Grade 1U ul 1 ul DEPC treated ddH 0 to 10 ul 6 Incubate at room temperature for 15 min 7 Incubate at 65 C for 15 min to inactivate the DNase I The absence of genomic DNA in the RNA sample can be verified by preparing a control reaction identical to the reaction on the following page using 2 U of Platinum Tag DNA polymerase Catalog no 10966 018 in place of the ThermoScript Plus Platinum Taq Enzyme Mix Continued on next page One Step Real Time RT PCR Continued One Step Real Time RT PCR Protocol The following protocol using Platinum Quantitative RT PCR ThermoScript One Step System has been optimized for LUX primers Because one step RT PCR is a complex reaction that is susceptible to artifacts further optimization may be required Note Keep all components reaction mixes and samples on ice After assembly tra
19. mplate for one step real time RT PCR is RNA usually total cellular RNA or mRNA The amount of template typically ranges from 1 pg to 100 ng per assay The purity and integrity of the RNA have a direct impact on results RNase and genomic DNA contamination are the most common problems and purification methods should be designed to avoid these We recommend using the Micro to Midi Total RNA Purification System Catalog no 12183 018 or TRIzol reagent Catalog no 15596 026 to isolate total RNA High quality total RNA can be purified from as little as 100 cells up to 10 cells or 200 mg of tissue To isolate mRNA we recommend using the FastTrack 2 0 mRNA Isolation Kit Catalog no K1593 02 The one step RT PCR enzyme mix should be optimized for real time PCR We recommend using the Platinum Quantitative RT PCR ThermoScript One Step System Catalog no 11731 015 which uses a ThermoScript Plus Platinum Tag enzyme mix It has been optimized for use in real time fluorescent PCR systems See the sample reaction on page 13 For optimal PCR conditions primer titrations of 50 500 nM per primer are recommended The 50 ul sample reaction on page 13 uses 200 nM of each primer equivalent to 1 ul of a 10 uM primer solution The optimal Mg concentration for a given target primer polymerase combination can vary between 1 mM and 10 mM but is usually in the range of 3 mM see the sample reaction on page 13 The o
20. n add an appropriate volume of TE buffer or ultrapure water close the tube rehydrate for 5 minutes and vortex for 15 seconds We recommend that you rehydrate primers at concentrations greater than 10 uM To prepare a 100 uM primer stock solution multiply the primer amount in nmoles by ten to determine the volume of diluent in ul After reconstitution store the primer stock at 20 C in the dark where it will be stable for 6 months or more Real Time PCR Introduction Template Specifications Primer Concentration Magnesium Concentration dNTP Concentration Instrument Specifications Instrument Settings This section provides guidelines and an optimized protocol for performing real time PCR using LUX primers The target template for real time PCR is linear single stranded or double stranded DNA cDNA or circular DNA such as plasmids The amount of DNA typically ranges from 10 to 10 copies or 1 pg to 10 ug of template See page 9 for instructions on generating cDNA using reverse transcription as part of two step real time RT PCR For optimal PCR conditions primer titrations of 50 500 nM per primer are recommended The 50 ul sample reaction on the following page uses 200 nM of each primer equivalent to 1 ul of a 10 uM primer solution The optimal Mg concentration for a given target primer polymerase combination can vary between 1 mM and 10 mM but is usually in the range of 3 mM
21. n hold 50 70 C 15 30 min hold PCR PCR 95 C 5 min hold 95 C 5 min hold 45 cycles of 45 cycles of 95 C 15 s 95 C 15 s 55 C 30 s 60 65 C 30 45 s 72 C 30 s 3 Add 40 ul of the Master Mix to an optical PCR tube or each well of a 96 well PCR plate 4 Add 10 ul of sample RNA to the tube or each well of the plate Cap or seal the tube plate 5 Gently mix and make sure that all components are at the bottom of the tube plate wells Centrifuge briefly if needed 6 Place reaction in the real time PCR instrument and run the program Collect and analyze results 13 Troubleshooting Problem Cause Solution Signal in controls with no DNA contamination template Ensure that amplification reactions are assembled in a DNA free environment Use of aerosol resistant barrier tips is recommended Take care to avoid cross contamination between primers or template DNA in different reactions Run PCR product on an agarose gel in an area separate from the reaction assembly area to confirm product Amplification of PCR carryover products Analyze PCR product on an agarose gel in an area separate from the reaction assembly area Use Platinum Quantitative PCR SuperMix UDG as specified in the sample protocol on page 7 Since dUTP is substituted for dTTP in the reaction cocktail any amplified DNA will contain uracil UDG prevents reamplification of PCR carryover products by removing uracil residues from si
22. ng a hot start enzyme for the RT reaction such as SuperScript II RT The following sample protocol uses the SuperScript First Strand Synthesis System for RT PCR Catalog no 11904 018 available from Invitrogen which includes all components needed for the first strand synthesis reaction except the RNA We recommend that you decrease the genomic DNA content in the RNA sample by performing a digest with DNase I Amplification Grade Catalog no 10868 015 as described below The DNase I digest is designed for up to 1 ug of RNA for larger amounts of RNA increase volumes accordingly Combine the following in a tube on ice Component Conc Volume RNA template z x pl DNase reaction buffer 10X 1 ul DNase I Amplification Grade 1U ul 1 ul DEPC treated ddH 0 to 10 ul 1 Incubate at room temperature for 15 min 2 Incubate at 65 C for 15 min to inactivate the DNase I Continued on next page Two Step Real Time RT PCR Continued Reverse The following protocol using the SuperScript First Strand Synthesis System Transcription for RT PCR has been optimized for LUX primers Follow this protocol to Protocol generate cDNA which can then be used in real time PCR see pages 6 7 1 Combine the following kit components in a tube on ice For multiplex reactions a master mix without RNA may be prepared oligo dT 12 18 0 5 ug pl 0 5 ul Random hexamers 50 ng pl 0 5 ul RNA up to 1 ug x ul 10x Buffer 2 ul 25 mM
23. ngle or double stranded DNA dU containing DNA that has been digested with UDG is unable to serve as template in future PCRs UDG is inactivated at high temperature during PCR thermal cycling thereby allowing amplification of genuine target sequence s Primer dimers Perform melting curve analysis of the PCR product identify dimers by lower melting point temperature Confirm that primer designs have low penalty scores 0 0 4 0 to minimize self annealing Redesign primers if necessary When redesigning primers note that you can first try redesigning only the unlabeled primer to save the cost of the LUX primer No or low signal Instruments setting not optimal Confirm that the cycling parameters are correct the quencher is set to none and the reference dye setting is correct Primer template sequences do not match Confirm that the sequences match Primer designs are not optimal Confirm that the primer design penalty scores are within the 0 0 4 0 range and the optimal melting temperatures have been specified Redesign primers if necessary When redesigning primers note that you can first try redesigning only the unlabeled primer to save the cost of the LUX primer Poor standard curve and Reaction is not optimized dynamic range Reoptimize reaction conditions Prepare primer titrations if necessary Reference dye not used Use ROX reference dye as specified 14 Accessory Products
24. nsfer the reaction to a thermal cycler preheated to the desired cDNA synthesis temperature 50 70 C and immediately begin RT PCR We recommend performing the cDNA synthesis reaction at 50 C but higher temperatures up to 70 C may be required for high GC content templates RNase inhibitor proteins such as RNaseOUT Catalog no 10777 019 may be added to the reaction following the addition of the 2X ThermoScript reaction mix to safeguard against degradation of RNA 1 To reduce well to well variation prepare a Master Mix of all the reaction ingredients except RNA The following table provides Master Mix volumes for one reaction and 50 reactions scale up or down as needed Component Stock conc Vol 1 rxn Vol 50 rxns Rxn conc ThermoScript Reaction Mix 2X 25 ul 1250 ul 1X ROX reference dye 50X Tul 50 ul 1X ThermoScript Plus Platinum Taq Enzyme Mix 50X Tul 50 pl 1X Reverse primer 10 uM Tul 50 ul 200 nM Forward primer 10 uM Tul 50 ul 200 nM Sterile distilled H O 11 yl 600 ul Total volume 40 ul 2000 ul Supplied at 2X concentration includes 400 uM of each dNTP and 6 mM MgSO or use DNase RNase Free distilled water Cat No 10977 015 2 Program the instrument with one of the following thermal cycling protocols for cDNA synthesis use a 30 min incubation at 50 C as a starting point 3 Step Cycling recommended 2 Step Cycling optional cDNA synthesis cDNA synthesis 50 70 C 15 30 mi
25. ptimal concentration of dATP dCTP dGTP and dTTP is 200 uM each If dUTP is used in place of dTTP its optimal concentration is 400 uM Continued on next page 11 One Step Real Time RT PCR Continued Instrument Specifications Instrument Settings Removing Genomic DNA from RNA Samples 12 LUX primers are compatible with a wide variety of real time PCR instruments with various detection capabilities including but not limited to the ABI PRISM 7700 7000 7900 and GeneAmp 5700 the Bio Rad iCycler the Stratagene Mx4000 and the Cepheid Smart Cycler At a minimum the instrument used to perform one step real time RT PCR with LUX primers must be able to e Detect fluorescence at each PCR cycle e Excite FAM and JOE labeled primers near their excitation wavelength maximums of 490 nm and 520 nm respectively TM e Detect the emission of FAM and JOE labeled LUX primers near their emission maximums of 520 nm and 550 nm respectively Please refer to the specific instrument s user manual for operating instructions Please follow the manufacturer s instructions for configuring your real time PCR instrument for use with LUX primers Use the following settings for performing one step real time RT PCR e Because LUX primers do not contain a quencher the quencher setting should be set to no quencher e We recommend the use of ROX reference dye Catalog no 12223 023 for normalization of wel
26. r your gene of interest and the JOE label for the housekeeping gene that you will use as the internal control After you have selected the label and synthesis scale you can submit your order directly from the LUX Designer Web site Each primer order will be shipped directly from Invitrogen s Custom Primer Facilities Labeled and unlabeled primers are shipped separately Labeled primers are shipped in an amber tube unlabeled primers are shipped in a clear tube Each primer ordered from Invitrogen s Custom Primer Facilities comes with a Certificate of Analysis COA verifying the amount and sequence LUX primers are provided in 50 nM or 200 nM synthesis scale They are tested post synthesis by optical density OD ratio measurements and mass spectroscopy to ensure efficient dye labeling and correct molecular weight and composition See the Certificate of Analysis shipped with each primer for more information Storing and Reconstituting Primers Primer Storage and Stability Reconstituting Primers Store primers at 20 C in the dark Primers are stable for e gt 12 months when stored at 20 C in lyophilized form e gt 6 months when stored at 20 C in solution Stability can be extended by storing at 70 C LUX primers are provided in 50 nmole or 200 nmole synthesis scale To reconstitute primers centrifuge the tube for a few seconds to collect the oligonucleotide in the bottom of the tube Carefully ope
27. therwise use this product or its components or materials made using this product or its components for commercial purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for the commercial purposes of the buyer and that such collaborator agrees in writing a to not transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for commercial purposes Commercial purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research Invitrogen Corporation will not assert a claim against the buyer of infringement of patents owned by Invitrogen 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 its components was employed provided that neither this product nor any of its components was used in the manufacture of such product If the purchaser is not willing to accept the limitations o
28. ture of oligodeoxyribonucleotides on the fluorescent properties of conjugated dyes Nucl Acids Res 30 2089 2195 Nazarenko I A Bhatnagar S K and Hohman R J 1997 A closed tube format for amplification and detection of DNA based on energy transfer Nucleic Acids Res 25 2516 2521 Nuovo G J Hohman R J Nardone G A and Nazarenko I 1999 In situ amplification using universal energy transfer labeled primers J Histochem Cytochem 47 273 279 Todd A V Fuery C J Impey H L Applegate T L and Haughton M A 2000 DzyNA PCR use of DNAzymes to detect and quantify nucleic acid sequences in a real time fluorescent format Clin Chem 46 625 630 Tyagi S and Kramer F R 1996 Molecular beacons probes that fluoresce upon hybridization Nature Biotechnol 14 303 308 Wittwer C T Herrmann M G Moss A A Rasmussen R P 1997 Continuous fluorescence monitoring of rapid cycle DNA amplification BioTechniques 22 130 138 2002 Invitrogen Corporation All rights reserved 19 o o 2 Invitrogen life technologies United States Headquarters Invitrogen Corporation 1600 Faraday Avenue Carlsbad California 92008 Tel 1760 603 7200 Tel Toll Free 1800 955 6288 Fax 1760 603 7229 Email tech_service invitrogen com European Headquarters Invitrogen Ltd 3 Fountain Drive Inchinnan Business Park Paisley PA4 9RF UK Tel Free Phone Orders 0800 269 210 Tel General Enquiri
29. uchi R Fockler C Dollinger G and Watson R 1993 Kinetic PCR analysis real time monitoring of DNA amplification reactions Biotechnology 11 1026 1030 Holland et al 1991 Detection of specific polymerase chain reaction product by utilizing the 5 3 exonuclease activity of Thermus aquaticus DNA polymerase Proc Natl Acad Sci USA 88 7276 7280 Kaboev O K Luchkina L A Tret iakov A N and Bahrmand A R 2000 PCR hot start using primers with the structure of molecular beacons hairpin like structure Nucleic Acids Res 28 e94 Knemeyer J P Marme N and Sauer M 2000 Probes for detection of specific DNA sequences at the single molecule level Anal Chem 72 3717 3724 Murchie A I H Clegg R M von Kitzing E Duckkett D R Diekmann S and Lilley D M J 1989 Fluorescence energy transfer shows that the four way DNA junction is a right handed cross of antiparallel molecules Nature 341 763 766 Myakishev M V Khripin Y Hu S and Hamer D H 2001 High throughput SNP genotyping by allele specific PCR with universal energy transfer labeled primers Genome Res 11 163 169 Nazarenko I Lowe B Darfler M Ikonomi P Schuster D and Rashtchian A 2002 Multiplex quantitative PCR using self quenched primers labeled with a single fluorophore Nucl Acids Res 30 e37 Nazarenko I Pires R Lowe B Obaidy M and Rashtchian A 2002 Effect of primary and secondary struc
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