LUX Fluorogenic Primers - Thermo Fisher Scientific
Contents
1. Invitrogen life technologies Instruction Manual LUX Fluorogenic Primers For real time PCR and RT PCR Version F 13 September 2004 25 0546 ii Table of Contents Tasse 10 ei e MS e De D Torr T rere ry tony Dee Tene eM Tonner Ry EEEE MOT ere nD etOr ore emTT Ty oT one 1 Designing and Ordering Custom LUX Primes 3 Storing and Reconstituting Pnirmers sse 5 Certified LUX Primer Sets for Housekeeping Genes 6 RealTime PGR etirdi neeaaea a a a e aa ae alter a a aE eid 7 Multiplex Real Time WCR 11 Two Step Real Time QR T PCR vi ssc tact cave cccscapcinadicned dares neared artted bidet Qetead itiBadadissmatads 12 One Step Real Time qRT PCR eee 16 TOUR ISS INO LNG cos oe e a sh a aa Ea aE ss a at aes eee EEEE 21 Accessory Products sss 23 Purchaser Notification sss eee 24 Technical SERVICE so iie e e eea ae e e ents a e e aea a outst E Eee Eaa EE a 25 Referente Se eee aa EE Ea a aE E a O re 27 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 qPCR and RT PCR qRT PCR Each primer pair in the LUX system includes a fluorogenic primer with a fluorophore attached to its 3 end and a corresponding unlabeled primer The fluorogenic primer has a short sequence tail of 4 6 nucleotides on the 5 end that is complementary to the 3 end of the2. The following protocol uses Platinum Quantitative PCR SuperMix UDG and has been optimized for the Roche LightCycler Consult the LightCycler documentation for detailed instructions on preparing the capillary tubes and operating the instrument FAM labeled LUX Primers are also compatible with Roche enzyme mixes TM Note JOE labeled LUX Primers are not compatible with the current version of the LightCycler use FAM labeled primers only The following protocol uses a 20 ul reaction volume Before proceeding see the real time qPCR guidelines on the previous pages 1 To reduce well to well variation prepare a Master Mix of all the reaction ingredients except template The following table provides volumes for one reaction and 34 reactions scale as needed Component Vol 1 rxn Vol 34 rxns Platinum Quantitative PCR SuperMix UDG 10 ul 340 ul FAM labeled LUX Primer 10 uM 1 u 34 ul Unlabeled primer 10 uM 1 ul 34 ul Bovine serum albumin 5 mg ml 1 u 34 ul Platinum Tad DNA Polymerase 0 12 ul 4ul Sterile distilled water to 18 ul to 612 pl Final concentration 0 03 U l Platinum Taq DNA polymerase 20 mM Tris HCl pH 8 4 50 mM KCI 3 mM MgCh 200 uM dGTP 200 uM dATP 200 uM dCTP 400 uM dUTP 1 U UDG Validated with non acetylated Ultrapure BSA 10 solution from Panvera Cat nos P2489 and P2046 5Total units of Platinum Taq DNA Polymerase in the reaction is 1 2 including 0 6 U from Platinum Quan
3. Placing the Order Product Qualification After you have selected a primer set labeled and unlabeled for a particular sequence you can specify the particular label and synthesis scale Custom LUX Primers are provided in 50 nM or 200 nM synthesis scale When selecting labels in a multiplex reaction we recommend using the FAM label for your gene of interest and the JOE label for the housekeeping gene that you will use as the internal control Certified LUX Primer Sets for Housekeeping Genes are recommended for the JOE labeled control gene After you have selected the label and synthesis scale you can submit your order to Invitrogen using the Web site or by e mail or fax Each primer order will be shipped directly from Invitrogen s Custom Primer Facilities Labeled primers are supplied in an amber tube unlabeled primers are supplied 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 Custom LUX Primers 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 Store primers at 20 C in the dark LUX Primers are stable for Stability Important Reconst
4. 0 06 U l Platinum Taq DNA polymerase 20 mM Tris HCl pH 8 4 50 mM KCI 3 mM MgCh 200 uM dGTP 200 uM dATP 200 uM dCTP 400 uM dUTP 0 04 U pl UDG 2 Program the real time qPCR instrument as follows Thermal Cycling 50 C 2 min hold UDG treatment 95 C 2 min hold 45 cycles of 95 C 15 s 55 C 30 s 72 C 30s Melting Curve Analysis Refer to instrument documentation 3 Add 45 ul of the Master Mix to an optical PCR tube or each well of a 96 well PCR plate 4 Add 5 ul 10 to 10 copies or 1 pg to 10 ug of the cDNA from the first strand synthesis reaction step 5 page 13 to each reaction vessel 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 qPCR instrument and run the program Collect and analyze results Continued on next page Two Step Real Time qRT PCR Continued Protocol for the Roche LightCycler The following protocol uses components from the SuperScript II Platinum Two Step qRT PCR Kit Catalog nos 11734 050 and 11734 068 and has been optimized for the Roche LightCycler Consult the LightCycler documentation for detailed instructions on preparing the capillary tubes and operating the instrument FAM labeled LUX Primers are also compatible with Roche enzyme mixes TM Note JOE labeled LUX Primers are not compatible with the current version
5. 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 structure of oligodeoxyribonucleotides on the fluorescent properties of conjugated dyes Nucl Acids Res 30 2089 2095 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 Tya
6. includes 0 4 mM of each dNTP and 6 mM MgSO See the Important note on primer concentration on page 16 2 Program the instrument with the following thermal cycling protocol for cDNA synthesis use a 15 min incubation at 50 C as a starting point cDNA synthesis 50 C for 15 min hold PCR 95 C for 2 min hold 40 50 cycles of 95 C 15 s 60 C 30 s Melting Curve Analysis optional Program according to instrument instructions 3 For each reaction add 40 ul of the master mix to a 0 2 ml microcentrifuge tube or each well of a 96 well PCR plate on ice 4 Add 10 pl of sample RNA 1 pg to 1 ug total RNA to each tube plate well and cap or seal 5 Gently mix and make sure that all components are at the bottom of the tube plate wells Centrifuge briefly if needed 6 Place reactions in a preheated thermal cycler programmed as described above Collect data and analyze results Continued on next page 19 One Step Real Time qRT PCR Continued Protocol for the Roche LightCycler The following protocol using the SuperScript III Platinum One Step Quantitative RT PCR System has been optimized for LUX Primers and the Roche LightCycler Further optimization may be required FAM labeled LUX Primers are also compatible with Roche enzyme mixes Note JOE labeled primers are not compatible with the current version of the LightCycler use FAM labeled primers only After assembly transfer the reaction to a t
7. primer The resulting hairpin secondary structure provides optimal quenching of the fluorophore see the figure below When the primer is incorporated into double stranded DNA during PCR the fluorophore is dequenched and the signal increases by up to 10 fold TM LUX Primers combine high specificity with simple design and streamlined protocols LUX Primers require no special probes or quenchers and are compatible with melting curve analysis of real time qPCR products which allows for the differentiation of amplicons and primer dimer artifacts by their melting temperatures LUX Primers are available with two different reporter dyes which provides for multiplexing capability You can custom design LUX Primers from a target DNA sequence using Web or desktop based software or order predesigned and validated Certified LUX Primer Sets for Housekeeping Genes Relative fluorescence 0 1 Hairpin primer L ee 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 Continued on next page Introduction Continued Applications Instrument Compatibility LUX Primers can be used in real time quantitative 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 Th
8. 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 Onthe home page go to the left hand column under 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 oO fF YN When you are finished entering information click the Submit button Your MSDS will be sent within 24 hours Continued on next page 25 Technical Service Continued Limited Warranty 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 wi
9. CDNA synthesis 95 C 0 s 95 C 2 min hold 55 C 15 sec 50 cycles of 95 C 0 increase 0 1 C s with 95 C 5s continuous acquisition 55 C 10 s single acquire 40 C 0s 72 C 10s Add 18 ul of Master Mix to each capillary tube of the LightCycler on ice Add 2 ul of sample RNA 1 pg to 1 ug total RNA to each capillary tube and cap the tube Centrifuge the tubes at 700 x g for 5 seconds Place the reaction tubes in the rotor of the LightCycler and run the program Collect and analyze results Troubleshooting Problem Cause Solution Signals are present in no template controls and or multiple peaks are present in the melting curve graph Template or reagents are contaminated by nucleic acids DNA cDNA Use melting curve analysis and or run the PCR products on a 4 agarose gel in an area separate from the reaction assembly area to identify contaminants To reduce the risk of contamination take standard precautions when preparing your PCR reactions Ideally amplification reactions should be assembled in a DNA free environment We recommend using aerosol resistant barrier tips Amplification of PCR carryover products Analyze the PCR product on a 4 agarose gel in an area separate from the reaction assembly area to identify contaminants We recommend using a UDG based carryover prevention system such as Platinum Quantitative PCR SuperMix UDG or the SuperScript III Platinum Two
10. Genes functionally validated to provide accurate reproducible results using standard LUX protocols They are supplied ready to use in TE buffer Each Certified LUX Primer Set includes a FAM or JOE labeled LUX primer and a corresponding unlabeled primer Each primer labeled and unlabeled is supplied at 100 ul and a concentration of 10 uM Available sets are listed below For additional information visit www invitrogen com LUX GenBank Forward Cat no Cat no Relative CDS PCR Product Product Accession no Reverse Label FAM label JOE label expression Location Size Range Human genes 18S rRNA X03205 Forward 115HM 01 115HM 02 n a 101 150 bp h6 ACTIN NM_001101 Forward 101H 01 101H 02 Exons 2 3 101 150 bp hATPSase NM_001686 Forward 108H 01 108H 02 4 n a 101 150 bp hB2M NM_004048 Forward 113H 01 113H 02 Exons 1 2 101 150 bp hGAPDH NM_002046 Forward 100H 01 100H 02 Exons 4 5 151 200 bp hPGK1 NM_000291 Forward 109H 01 109H 02 4 n a 50 100 bp hPPIA NM_021130 Forward 106H 01 106H 02 Exons 2 3 50 100 bp hRPL4 NM_000968 Reverse 103H 01 103H 02 4 Exons 8 9 101 150 bp hEEF1G NM_001404 Forward 107H 01 107H 02 n a 50 100 bp hHPRT1 NM_000194 Reverse 105H 01 105H 02 Exons 5 6 50 100 bp hSDHA NM_004168 Forward 102H 01 102H 02 4 Exons 12 13 50 100 bp hTFRC NM_003234 Forward 111H 01 111H 02 Exons 10 11 101 150 bp hGUS NM_000181 Forward 112H 01 112H 02 Exons 7 8 101 150 bp hHMBS NM_000190 Forwar
11. Inc iCycler Mx4000 Mx3000 Rotor Gene and Smart Cycler are trademarks of their respective companies Designing and Ordering Custom LUX Primers LUX Designer To design and order custom LUX Primers for your genes of interest visit the Primer Design Invitrogen LUX Web site at www invitrogen com LUX and follow the link to Software the LUX Designer software The software is available as either a Web based application or a Microsoft Windows compatible download Follow the step by step instructions in the software to submit your target sequence and generate primer designs TM 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 review them select a design select the fluorophore labels and place your order Guidelines for When you submit a target sequence containing your gene of interest keep in Submitting a mind the following design criteria Target Sequence e The optimal amplicon length for real time qPCR ranges from 80 to 200 bases You can specify a minimum optimal and maximum amplicon length when y
12. Master Mix volumes for one reaction and 50 reactions scale up or down as needed Component Vol 1 rxn Vol 50 rxns Platinum Quantitative PCR SuperMix UDG 25 ul 1250 ul ROX Reference Dye optional 1 yul 50 pl Labeled LUX Primer 10 uM Tul 50 ul Unlabeled primer 10 uM Tul 50 ul Sterile distilled water to 40 ul to 2000 ul Final concentration 0 03 U l Platinum Taq DNA polymerase 20 mM Tris HCl pH 8 4 50 mM KCI 3 mM MgCh 200 uM dGTP 200 uM dATP 200 uM dCTP 400 uM dUTP 1 U UDG 2or use DNase RNase Free Distilled Water Cat No 10977 015 2 Program the real time qPCR instrument as follows 3 Step Cycling recommended 2 Step Cycling optional 50 C 2 min hold UDG treatment 50 C 2 min hold UDG treatment 95 C 2 min hold 95 C 2 min hold 45 cycles of 45 cycles of 95 C 15s 95 C 15 s 55 C 30 s 60 65 C 30 45 s 72 C 30 s Melting Curve Analysis recommended Refer to instrument documentation 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 pl of template in TE or sterile dH2O to each reaction vessel 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 qPCR instrument and run the program Collect and analyze results Continued on next page Real Time qPCR Continued Protocol for the Roche LightCycler
13. mM see the sample reaction on page 19 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 Continued on next page One Step Real Time qRT PCR Continued ROX Reference Dye Bovine Serum Albumin BSA Melting Curve Analysis We recommend using ROX Reference Dye Cat no 12223 023 to normalize the fluorescent reporter signal for instruments that are compatible with this option ROX Reference Dye can be used to adjust for non PCR related fluctuations in fluorescence between reactions and provides a stable baseline in multiplex reactions It is composed of a glycine conjugate of 5 carboxy X rhodamine succinimidy ester 25 uM in 20 mM Tris HCl pH 8 4 0 1 mM EDTA and 0 01 Tween 20 ROX is supplied at 50X concentration Add 1 ul of ROX for every 50 ul of reaction volume Bovine serum albumin BSA is required in qPCR reactions on the Roche LightCycler because the glass capillaries in the LightCycler have a high surface to volume ratio and the glass surface binds molecules like Taq DNA polymerase effectively removing them from the reaction The addition of BSA blocks this surface binding Nonacetylated BSA is strongly recommended because acetylated BSA will inhibit PCR at the concentrations required in LightCycler reactions This inhibition is most likely due to the transfer of acetyl groups to essential components
14. of the LightCycler use FAM labeled primers only The following protocol uses a 20 ul reaction volume Before proceeding see the real time qPCR guidelines on pages 7 8 1 To reduce well to well variation prepare a Master Mix of all the reaction ingredients except template The following table provides volumes for one reaction and 34 reactions scale as needed Component Vol 1 rxn Vol 34 rxns Platinum Quantitative PCR SuperMix UDG 10 ul 340 ul FAM labeled LUX Primer 10 uM Tul 34 ul Unlabeled primer 10 uM Tul 34 ul BSA UltraPure 5 mg ml 1 u 34 ul Platinum Tag DNA Polymerase 0 12 ul 4 ul Sterile distilled water to 18 ul to 612 ul 1 Final concentration 0 06 U l Platinum Taq DNA polymerase 20 mM Tris HCl pH 8 4 50 mM KCI 3 mM MgCh 200 uM dGTP 200 uM dATP 200 uM dCTP 400 uM dUTP 0 04 U pl UDG Total units of Platinum Taq DNA Polymerase in the reaction is 1 2 including 0 6 U from Platinum Quantitative PCR SuperMix UDG 2 Set the fluorescence on the Roche LightCycler to the F1 channel 3 Program the instrument as follows Thermal Cycling Melting Curve Analysis optional Program choice Amplification Program choice Melting curve Analysis mode Quantification Analysis mode Melting curves Cycling Cycling 50 C 2 min hold UDG treatment 95 C 0s 95 C 2 min hold 55 C 15 sec 45 cycles of 95 C 0 increase 0 1 C s with 94 C 5s continuous acquisition 55 C 10 s single acquire 40 C
15. 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 of 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
16. sensitivity 1 Combine the following kit components in a tube on ice For multiple reactions a master mix without RNA may be prepared 2X RT Reaction Mix 10 ul RT Enzyme Mix 2 ul RNA 10 pg to 1 ug x pl DEPC treated water to 20 ul Gently mix tube contents and incubate at 25 C for 10 min Incubate tube at 42 C for 50 min Terminate the reaction at 85 C at 5 min and then chill on ice Add 1 ul 2 U of E coli RNase H and incubate at 37 C for 20 min Store the reaction at 20 C until use Proceed to the real time qPCR protocol on the following page S e R be Continued on next page 13 Two Step Real Time qRT PCR Continued Real Time qPCR Protocol for Instruments Using PCR Tubes or Plates The following real time qPCR protocol uses components from the SuperScript III Platinum Two Step qRT PCR Kit Catalog nos 11734 050 and 11734 068 It has been optimized for use with real time qPCR instruments that use tubes or plates See the guidelines on pages 7 8 A protocol for the Roche LightCycler is provided on the following page 1 To reduce well to well variation prepare a Master Mix of all the reaction ingredients except template Component Vol 1 rxn Vol 50 rxns Platinum Quantitative PCR SuperMix UDG 25 ul 1250 ul ROX Reference Dye optional 1 yul 50 pl Labeled LUX Primer 10 uM 1 ul 50 ul Unlabeled primer 10 uM 1 u 50 ul Sterile distilled water to 45 ul to 2250 ul Final concentration
17. 0s 72 C 10s 4 Add 18 pl of Master Mix to each capillary tube of the LightCycler 5 Add 2 pl 10 to 10 copies or 1 pg to 10 ug of the cDNA from the first strand synthesis reaction step 5 page 13 to each tube and cap the tube 6 Centrifuge the tubes at 700 x g for 5 seconds 7 Place the reaction tubes in the rotor of the LightCycler and run the program Collect and analyze results 15 One Step Real Time qRT PCR Introduction Primer Concentration Template Specifications Enzyme Specifications Magnesium Concentration dNTP Concentration In one step real time qRT PCR cDNA synthesis and PCR are performed in a single reaction tube using total RNA or mRNA as starting material The optimized enzyme mixture includes both a reverse transcriptase and a DNA polymerase This section provides guidelines and an example protocol for performing one step real time qRT PCR using LUX Primers The example protocol uses the SuperScript II Platinum One Step Quantitative RT PCR System for superior specificity and sensitivity with LUX Primers For optimal one step qRT PCR primer titrations of 50 500 nM per primer are recommended The 50 ul sample reaction on page 19 uses 200 nM of each primer equivalent to 1 ul of a 10 uM primer solution Also see the note below In one step qRT PCR the reverse primer drives the reverse transcription reaction We have found that doubling the concentration of the
18. 19 20 using 2 U of Platinum Taq DNA polymerase Catalog no 10966 018 in place of the SuperScript ITI RT Platinum Taq Mix Continued on next page One Step Real Time qRT PCR Continued Protocol for Instruments Using PCR Tubes or Plates The following protocol using the SuperScript III Platinum One Step Quantitative RT PCR System has been optimized for LUX Primers Further optimization may be required Note Keep all components reaction mixes and samples on ice After assembly transfer the reaction to a thermal cycler preheated to the cDNA synthesis temperature and immediately begin RT PCR We recommend performing the cDNA synthesis reaction at 50 C but higher temperatures up to 60 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 to safeguard against degradation of RNA 1 The following table provides Master Mix volumes for a standard 50 pl reaction size Note that preparation of a master mix is crucial in quantitative applications to reduce pipetting errors Component Vol 1 rxn Vol 100 rxns SuperScript II RT Platinum Taq Mix 1 ul 100 ul 2X Reaction Mix 25 ul 2500 pl ROX Reference Dye optional 1 ul 100 ul Labeled LUX Primer 10 uM 1 u 100 ul Unlabeled primer 10 uM 1 ul 100 ul RNaseOUT optional 1 u 100 ul Sterile distilled water to 40 ul to 4000 ul Supplied at 2X concentration
19. M For first strand cDNA synthesis alone we recommend the SuperScript II First Strand Synthesis System for RT PCR Catalog no 18080 051 Continued on next page Two Step Real Time qRT PCR Continued Removing Genomic DNA from RNA Samples Reverse Transcription Protocol We strongly recommend that you decrease the genomic DNA content in the RNA sample by performing a digest with DNase I Amplification Grade Catalog no 18068 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 ul DNase reaction buffer 10X 1 u DNase I Amplification Grade 1U ul Tul DEPC treated ddH 0 to 10 ul 1 Incubate at room temperature for 15 min 2 Add 1 ul of 25 mM EDTA solution to the reaction mixture and incubate at 65 C for 10 min to inactivate the DNase I The following protocol for generating first strand cDNA uses components from the SuperScript III Platinum Two Step qRT PCR Kit Catalog nos 11734 050 and 11734 068 The RT Enzyme Mix contains SuperScript II RT and RNaseOUT The 2X RT Reaction Mix contains oligo dT 2 2 5 uM random hexamers 2 5 ng ul 10 mM MgCh and dNTPs Note The E coli RNase H digestion step is included to remove the RNA template from the cDNA RNA hybrid molecule after first strand synthesis This has been shown to increase PCR
20. Step qRT PCR Kit protocols provided on pages 9 10 and pages 12 15 respectively 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 single 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 or other primer artifacts are present Use melting curve analysis of the PCR product to identify primer dimers by their lower melting point temperature Confirm that your primer designs have low scores 0 0 4 0 when generated by the LUX Designer to minimize self annealing Redesign primers if necessary If you are redesigning primers you can first try redesigning only the unlabeled primer to save the cost of the LUX primer Primer contamination or truncated or degraded primers can also lead to artifacts Check the purity of your primers by gel electrophoresis If agarose gels are used we recommend cooling the gels before visualization with intercalating dyes Continued on next page 21 Troubleshooting Continued Problem Cause Solution No amplification curve appears on the qPCR graph There is no PCR product Run the PCR product on a gel to determine whether PCR wor
21. d 110H 01 110H 02 Exons 2 3 50 100 bp hTBP NM_003194 Forward 104H 01 104H 02 Exons 3 4 101 150 bp hUBC NM_021009 Forward 114H 01 114H 02 4 n a 50 100 bp Mouse rat genes 18S rRNA X03205 Forward 115HM 01 115HM 02 n a 101 150 bp mf ACTIN NM_007393 Forward 101M 01 101M 02 Exons 2 3 101 150 bp mB2M X01838 Forward 113M 01 113M 02 n a 50 100 bp mEEF1G AF321126 Forward 107M 01 107M 02 n a 101 150 bp mGAPDH NM_008084 Forward 100M 01 100M 02 Exons 4 5 151 200 bp mPGK1 NM_008828 Forward 109M 01 109M 02 Exons 1 2 101 150 bp mPPIA NM_008907 Reverse 106M 01 106M 02 Exons 1 2 50 100 bp mRPL4 NM_022510 Forward 103M 01 103M 02 Exons 2 3 151 200 bp mHPRT1 NM_013556 Forward 105M 01 105M 02 Exons 6 7 50 100 bp mSDHA AF095938 Forward 102M 01 102M 02 Exons 6 7 50 100 bp mATPSase NM_016774 Forward 108M 01 108M 02 n a 50 100 bp mGUS NM_010368 Forward 112M 01 112M 02 Exons 7 8 50 100 bp mHMBS XM_129404 Reverse 110M 01 110M 02 Exons 4 5 50 100 bp mTBP NM_013684 Forward 104M 01 104M 02 Exons 3 4 101 150 bp mTFRC NM_011638 Forward 111M 01 111M 02 Exons 2 3 101 150 bp Drosophila genes d18S rRNA AY037174 Reverse 115D 01 115D 02 n a 101 150 bp dActin NM_079486 Forward 101D 01 101D 02 Exons 2 3 50 100 bp Real Time qPCR Introduction Template Specifications Primer Concentration Magnesium Concentration dNTP Concentration DNA Polymera
22. d 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 Higuchi R Fockler C Walsh P S and Griffith R 1992 Simultaneous amplification and detection of specific DNA sequences Biotechnology 10 413 417 Higuchi 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
23. eping gene used as an internal control to normalize between different reactions We recommend using Certified LUX Primer Sets for Housekeeping Genes for the internal control see page 6 Note We recommend selecting a housekeeping gene that matches the relative expression level of your gene of interest The relative expression levels of predesigned certified LUX Primer Sets are shown on page 6 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 Component Volume Platinum Quantitative PCR SuperMix UDG 2X 25 pl ROX Reference Dye 50X 1 ul Template 10 ul Forward primer 1 FAM label 10 uM 1 u Reverse primer 1 10 uM 1 u Forward primer 2 JOE label 10 uM 1 ul Reverse primer 2 10 uM 1 ul Sterile distilled water to 50 pl All other reaction volumes remain the same Follow the thermal cycling guidelines provided in Protocol for Instruments Using PCR Tubes or Plates on page 9 If you have difficulty performing the multiplex reaction using these guidelines see the optimization hints below If you notice a decline in PCR efficiency in your multiplex real time qPCR 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
24. ey have a broad dynamic range of 7 8 orders See the guidelines and sample protocols for qPCR on pages 7 10 and guidelines and sample protocols for qRT PCR on pages 12 20 Multiplex applications use separate FAM and JOE labeled primer sets to detect two different genes in the same sample Typically a custom designed FAM labeled primer set would be used to detect the gene of interest and a JOE labeled Certified LUX Primer Set would be used to detect a housekeeping gene as an internal control See the optimization guidelines for multiplex qPCR on page 11 LUX Primers are compatible with a wide variety of real time qPCR instruments including but not limited to the ABI PRISM 7700 7000 and 7900 and GeneAmp 5700 the Bio Rad iCycler the Stratagene Mx4000 and Mx3000 the Cepheid Smart Cycler the Corbett Research Rotor Gene and the Roche LightCycler At a minimum the real time qPCR instrument should e Detect fluorescence at each PCR cycle e Excite and detect FAM labeled LUX Primers near their excitation emission wavelengths of 490 520 nm and or s Excite and detect JOE labeled LUX Primers near their excitation emission wavelengths of 520 550 nm Refer to the specific instrument s user manual for operating instructions ABI PRISM is a registered trademark of Applera Corporation GeneAmp is a registered trademark of Roche Molecular Systems Inc LightCycler is a registered trademark of Idaho Technologies
25. fficiency between dilutions PCR efficiency is below 90 The PCR conditions are suboptimal Verify that the amount of primers you are using is correct and that the labeled primer has not been exposed to direct light Verify that the reagents you are using have not been freeze thawed multiple times and have not sat at room temperature for too long Reagent concentration in a multiplex reaction may be limiting the rate of the reaction Perform a single plex reaction using the same primers and template to check efficiency Then determine which one of your primer sets should be in limiting concentration See the multiplex guidelines on page 11 Accessory Products TM Products The following products are available for use with LUX Primers in real time qPCR and qRT PCR protocols Platinum Quantitative PCR SuperMix UDG 100 rxns 11730 017 500 rxns 11730 025 SuperScript III Platinum Two Step qRT PCR Kit 100 PCRs 11734 050 SuperScript III Platinum One Step Quantitative RT 100 rxns 11732 020 Platinum Taq DNA Polymerase 100 rxns 10966 018 250 rxns 10966 026 500 rxns 10966 034 5 000 rxns 10966 083 TRizol Reagent 100 ml 15596 026 A O o oom es ROX Reference Dye 500 ul 12223 023 DNase I Amplification Grade 1 U l 100 U 18068 015 RNaseOUT Recombinant Ribonuclease Inhibitor 5 000 U 10777 019 40 U ul 10 mM dNTP Mix 100 pl 18427 013 DEPC treated water 4x 1 25 ml 10813 012 23 P
26. 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 manuals 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
27. gi 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 2004 Invitrogen Corporation All rights reserved 27 gt 3 Invitrogen life technologies United States Headquarters Invitrogen Corporation 1600 Faraday Avenue Carlsbad California 92008 Tel 1 760 603 7200 Tel Toll Free 1 800 955 6288 Fax 1 760 603 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 Enquiries 0800 5345 5345 Fax 44 0 141 814 6287 Email eurotech invitrogen com International Offices Argentina 5411 4556 0844 Australia 1 800 331 627 Austria 0800 20 1087 Belgium 0800 14894 Brazil 0800 11 0575 Canada 800 263 6236 China 10 6849 2578 Denmark 80 30 17 40 France 0800 23 20 79 Germany 0800 083 0902 Hong Kong 2407 8450 India 11 577 3282 Italy 02 98 22 201 Japan 03 3663 7974 The Netherlands 0800 099 3310 New Zealand 0800 600 200 Norway 00800 5456 5456 Spain amp Portugal 900 181 461 Sweden 020 26 34 52 Switzerland 0800 848 800 Taiwan 2 2651 6156 UK 0800 838 380 For other countries see our website www invitrogen com
28. hermal cycler preheated to the cDNA synthesis temperature and immediately begin RT PCR We recommend performing the cDNA synthesis reaction at 50 C but higher temperatures up to 60 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 to safeguard against degradation of RNA 1 The following table provides Master Mix volumes for a standard 20 pl reaction size Note that preparation of a master mix is crucial in quantitative applications to reduce pipetting errors Component Vol 1 rxn Vol 34 rxns SuperScript II RT Platinum Tag Mix 0 8 ul 27 2 ul 2X Reaction Mix 10 ul 340 ul FAM labeled LUX Primer 10 M 1 u 34 ul Unlabeled primer 10 uM 1 ul 34 ul Bovine serum albumin 5 mg ml Tul 34 ul Sterile distilled water to 18 ul to 612 ul Includes 0 4 mM of each dNTP and 6 mM MgSO In the LightCycler reaction the LUX Fluorogenic Primer must be FAM labeled 3See the Important note on primer concentration on page 16 Validated with non acetylated Ultrapure BSA 10 solution from Panvera Cat nos P2489 and P2046 Set the fluorescence on the Roche LightCycler to the F1 channel Program the instrument as follows Thermal Cycling Melting Curve Analysis optional Program choice Amplification Program choice Melting curve Analysis mode Quantification Analysis mode Melting curves Cycling Cycling 45 C 30 min hold
29. ituting Primers s 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 TM Be careful to minimize the exposure of labeled LUX Primers to direct light as this can reduce their fluorescent intensity Custom LUX Primers are provided lyophilized 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 open 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 Certified LUX Primer Sets for Housekeeping Genes Certified LUX Primer Sets for Certified LUX Primer Sets for Housekeeping Genes are predesigned primer sets for genes that are commonly used as internal controls for normalizing Housekeeping real time qRT PCR experiments These primer sets have been optimized and
30. ked Then proceed to the troubleshooting steps below No PCR product is evident either in the qPCR graph or on a gel The protocol was not followed correctly Verify that all steps have been followed and the correct reagents dilutions volumes and cycling parameters have been used Template contains inhibitors nucleases or proteases or has otherwise been degraded Purify or re purify your template Primer designs are not optimal Confirm that you are using the correct primers for your sequence the primer design scores are within the 0 0 4 0 range in the LUX Designer 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 PCR product is evident in the gel but not on the qPCR graph qPCR instrument settings are incorrect Confirm that you are using the correct instrument settings dye selection reference dye filters acquisition points etc Problems with your specific qPCR instrument For instrument specific tips and troubleshooting using LUX Primers see the instrument protocols at www invitrogen com lux PCR efficiency is above 110 Template contains inhibitors nucleases or proteases or has otherwise been degraded Purify or re purify your template Inhibitors in the template may result in changes in PCR e
31. ll 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 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 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 incorporate
32. moving Genomic DNA from RNA Samples Follow the manufacturer s instructions for configuring your real time qPCR instrument for use with LUX Primers Note the following general settings e Program your instrument to perform melting curve analysis at the end of thermocycling if this option is available TM e The quencher setting on the instrument should reflect the fact that LUX Primers do not contain a quencher e We recommend using ROX Reference Dye if your instrument is compatible with this option Adjust the instrument settings accordingly Additional guidelines and settings for specific instruments are available at www invitrogen com lux click on Instrument Protocols We recommend that you decrease the genomic DNA content in the RNA sample by performing a digest with DNase I Amplification Grade Catalog no 18068 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 ul 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 Add 1 pl of 25 mM EDTA solution to the reaction mixture and incubate at 65 C for 10 min to inactivate the DNase I To verify the absence of genomic DNA in the RNA sample prepare a control reaction identical to the reactions on pages
33. n pages 7 8 The target template for two step qRT PCR is total RNA or mRNA High quality intact RNA is essential for full length high quality cDNA synthesis and accurate quantification Starting material can range from 10 pg to 1 ug total RNA Then use 1 pg to 10 ug of the cDNA from the first strand reaction in the qPCR step The purity and integrity of the starting 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 Isolation of mRNA is typically not required but can be performed using the FastTrack 2 0 mRNA Isolation Kit Catalog no K1593 02 For two step qRT PCR we recommend using a high specificity high yield reverse transcriptase such as SuperScript III Reverse Transcriptase and a hot start DNA polymerase such as Platinum Taq DNA Polymerase The SuperScript III Platinum Two Step qRT PCR Kit Catalog nos 11734 050 and 11734 068 includes SuperScript III RT Platinum Tag DNA Polymerase and all the other necessary components for two step qRT PCR except the RNA See the example protocol on page 13 T
34. of the PCR like the Tag DNA polymerase To ensure that the BSA does not contain RNA or DNA we recommend using ultrapure molecular biology grade nonacetylated BSA from Panvera Invitrogen Cat nos P2489 and P2046 Melting curve analysis is strongly recommended during one step qRT PCR to identify the presence of primer dimers and analyze the specificity of the reaction Program your real time instrument to perform this analysis after thermocycling Melting curve analysis identifies the change in fluorescent signal that occurs as double stranded DNA dsDNA dissociates or melts into single stranded DNA By identifying the temperature at which the dsDNA dissociates you can distinguish smaller artifacts like primer dimers with a lower annealing temperature from larger amplicons with a higher annealing temperature The presence of primer dimers in samples containing template decreases reaction efficiency and obscures analysis and determination of cycle thresholds The formation of primer dimers most often occurs in no template controls where the polymerase enzyme is essentially idle and in this case the quantitative analysis of the template samples is not affected Melting curve analysis of no template controls can discriminate between primer dimers and spurious amplification due to contaminating nucleic acids in reagent components Continued on next page 17 One Step Real Time qRT PCR Continued Instrument Settings Re
35. ou 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 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 PCR annealing temperatures from 55 to 64 C are appropriate When you first submit a sequence the Disable Score Based Rejection checkbox should not be checked the resulting scores provide an important measure of primer suitability Scores in the range of 0 0 4 0 are acceptable If no primers with a score of 4 0 or lower can be generated from a sequence you can disable score based rejection and redesign the primers See the LUX Designer Help for additional guidance TM Selecting a Primer After you submit your sequence LUX Designer will first generate one or Design more designs for the labeled primer The labeled primer can be either the forward or the reverse primer 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 Custom LUX Primers Continued Selecting Labels
36. reverse primer from 200 nM to 400 nM can in some cases decrease the cycle threshold for detecting a given target concentration and thus increase sensitivity See pages 3 4 for guidance on primer design The target template for one step real time qRT 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 qRT PCR enzyme mix should contain an optimized mixture of a high specificity high yield reverse transcriptase and a hot start DNA polymerase We recommend using the SuperScript IIT Platinum One Step Quantitative RT PCR System Catalog nos 11732 020 and 088 which uses a SuperScript III RT Platinum Tag enzyme mix See the sample reactions on pages 19 20 The optimal MgCl concentration for a given target primer polymerase combination can vary between 1 mM and 10 mM but is usually in the range of 3
37. s been specifically formulated to provide optimal performance in real time qPCR systems We recommend using ROX Reference Dye Cat no 12223 023 to normalize the fluorescent reporter signal in real time qPCR for instruments that are compatible with this option ROX Reference Dye can be used to adjust for non PCR related fluctuations in fluorescence between reactions and provides a stable baseline in multiplex reactions It is composed of a glycine conjugate of 5 carboxy X rhodamine succinimidyl ester 25 uM in 20 mM Tris HCl pH 8 4 0 1 mM EDTA and 0 01 Tween 20 ROX is supplied at 50X concentration Add 1 ul of ROX for every 50 ul of reaction volume For convenience and to reduce pipetting errors you can premix a solution of ROX and Platinum Quantitative PCR SuperMix UDG Add 1 ul of ROX for every 25 ul of SuperMix UDG Store mixture at either 20 C or 4 C in the dark Continued on next page Real Time qPCR Continued Bovine Serum Albumin BSA Melting Curve Analysis Instrument Settings Bovine serum albumin BSA is required in qPCR reactions on the Roche LightCycler because the glass capillaries in the LightCycler have a high surface to volume ratio and the glass surface binds molecules like Taq DNA polymerase effectively removing them from the reaction The addition of BSA blocks this surface binding Nonacetylated BSA is strongly recommended because acetylated BSA will inhibit PCR at the concentra
38. se ROX Reference Dye Real time qPCR uses genomic DNA cDNA or plasmid DNA as starting material This section provides guidelines and example protocols for TM performing real time qPCR using LUX Primers The target template for real time qPCR is linear single stranded or double stranded DNA cDNA or circular DNA such as plasmids that has been linearized The amount of DNA typically ranges from 10 to 10 copies or 1 pg to 10 ug of template See page 12 for instructions on generating cDNA using reverse transcription as part of two step real time qRT PCR For optimal PCR conditions primer titrations of 50 500 nM per primer are recommended The sample reactions on pages 9 10 use 200 nM of each primer equivalent to 1 ul of a 10 uM primer solution in a 50 ul reaction 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 reactions on pages 9 10 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 We recommend using a hot start DNA polymerase preferably one that has been optimized for real time qPCR Platinum Quantitative PCR SuperMix UDG Catalog no 11730 017 is a 2X concentrated ready to use mixture containing all components except primers and template It uses Platinum Taq DNA polymerase and ha
39. t components Follow the manufacturer s instructions for configuring your real time qPCR instrument for use with LUX Primers Note the following general settings e Program your instrument to perform melting curve analysis at the end of thermocycling if this option is available TM e The quencher setting on the instrument should reflect the fact that LUX Primers do not contain a quencher e We recommend using ROX Reference Dye if your instrument is compatible with this option Adjust the instrument settings accordingly Additional guidelines and settings for specific instruments are available at www invitrogen com lux click on Instrument Protocols Continued on next page Real Time qPCR Continued Protocol for Instruments Using PCR Tubes or Plates The following protocol uses Platinum Quantitative PCR SuperMix UDG with ROX reference reagent It has been optimized for use with real time qPCR instruments that use PCR tubes or plates A protocol for the Roche LightCycler is provided on the following page Note The following protocol uses a 50 ul reaction volume smaller volumes may be used depending on the requirements of your instrument Before proceeding see the real time qPCR guidelines on the previous pages For multiplex reactions see the guidelines on page 11 1 To reduce well to well variation prepare a Master Mix of all the reaction ingredients except template The following table provides
40. the primer concentration of the gene with the highest expression levels typically the housekeeping gene to 1 2 the primer concentration of the other gene For example in a standard 50 1 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 5 ul each 2 Increase the MgCl in the reaction from 3 mM to 6 mM 3 Double the amount of polymerase enzyme to 0 06 U l of reaction volume If 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 4 Increase the dNTP concentrations in the reaction to 400 uM each 11 Two Step Real Time qRT PCR Introduction Note Template Specifications Enzyme Specifications Real time qRT PCR uses RNA as starting material in a reverse transcription reaction to generate first strand cDNA The cDNA is then quantified in a separate real time qPCR reaction In two step qRT PCR first strand synthesis is performed and then the reaction is transferred to a separate tube for the qPCR reaction This section provides guidelines for two step qRT PCR and an optimized protocol using the SuperScript III Platinum Two Step qRT PCR Kit For the real time qPCR portion of the two step protocol see additional guidelines on primers magnesium dNTPs ROX Reference Dye BSA melting curve analysis and instrument settings o
41. tions required in LightCycler reactions This inhibition is most likely due to the transfer of acetyl groups to essential components of the PCR like the Tag DNA polymerase To ensure that the BSA does not contain RNA or DNA we recommend using ultrapure molecular biology grade nonacetylated BSA from Panvera Invitrogen Cat nos P2489 and P2046 Melting curve analysis is strongly recommended during qPCR to identify the presence of primer dimers and analyze the specificity of the reaction Program your real time instrument to perform this analysis after thermocycling Melting curve analysis identifies the change in fluorescent signal that occurs as double stranded DNA dsDNA dissociates or melts into single stranded DNA By identifying the temperature at which the dsDNA dissociates you can distinguish smaller artifacts like primer dimers with a lower annealing temperature from larger amplicons with a higher annealing temperature The presence of primer dimers in samples containing template decreases PCR efficiency and obscures analysis and determination of cycle thresholds The formation of primer dimers most often occurs in no template controls where the polymerase enzyme is essentially idle and in this case the quantitative analysis of the template samples is not affected Melting curve analysis of no template controls can discriminate between primer dimers and spurious amplification due to contaminating nucleic acids in reagen
42. titative PCR SuperMix UDG tor use DNase RNase Free Distilled Water Cat No 10977 015 2 Set the fluorescence on the Roche LightCycler to the F1 channel 3 Program the instrument as follows Thermal Cycling Melting Curve Analysis optional Program choice Amplification Program choice Melting curve Analysis mode Quantification Analysis mode Melting curves Cycling Cycling 50 C 2 min hold UDG treatment 95 C 0s 95 C 2 min hold 55 C 15 sec 45 cycles of 95 C 0 increase 0 1 C s with 94 C 5s continuous acquisition 55 C 10 s single acquire 40 C 0s 72 C 10s Add 18 ul of Master Mix to each capillary tube of the LightCycler Add 2 ul of template to each tube and cap the tube Centrifuge the tubes at 700 x g for 5 seconds Sy US ia Place the reaction tubes in the rotor of the LightCycler and run the program Collect and analyze results Multiplex Real Time qPCR Multiplex Real Time qPCR Optimizing Multiplex Conditions In multiplex real time qPCR different sets of primers with different fluorogenic labels are used to amplify separate genes in the template DNA Multiplexing with LUX Primers offers simplified PCR kinetics and increased reaction efficiency when compared with probe based technologies because only two oligos are used per target LUX Primers have been tested in multiplex reactions using a FAM labeled primer set for the gene of interest and a JOE labeled set for a houseke
43. urchaser 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 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 otherwise 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
Download Pdf Manuals
Related Search