pGL3 Luciferase Reporter Vectors Technical Manual #TM033

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When studying promoter functionalities, it is often desirable to include asecond reporter (e.g., Renilla luciferase) as an internal control for normalization.Plasmids derived from pGL3 or pGL4 vectors can be co-transfected with Renillaluciferase vectors, such as phRL-TK, and assayed using the Dual-Luciferase ®Reporter Assay System (Cat.# E1910) or the Dual-Glo Luciferase AssaySystem (Cat.# E2920).Table 2. Characteristics of Promega Dual-Luciferase Assays.Dual-Luciferase ®Dual-Glo AssayAssayFormat NH HProcess bench scale batchNumber of Steps 5 2Sensitivity higher lowerSignal Half-Life—firefly ~9 minutes ~2 hoursSignal Half-Life—Renilla ~2 minutes ~2 hoursPrecision High HighCell Lysis Time ~10 minutes ~15 minutesmaximummaximumNH = nonhomogeneous (first create a lysate); H = homogeneousVII. Sequencing of Luciferase Reporter VectorsYou may desire to sequence the DNA inserted into the Luciferase ReporterVectors. Two examples of such applications are to determine the exact positionof generated deletions and to confirm production of a site-specific mutation.Three primers are available for sequencing the pGL3 Vectors: RVprimer3(Reporter Vector Primer 3) for sequencing clockwise across the upstreamcloning sites, RVprimer4 for sequencing counterclockwise across the BamHIand SalI cloning sites downstream of luc+, and GLprimer2 for sequencingcounterclockwise upstream of luc+.RVprimer3RVprimer4GLprimer25´-CTAGCAAAATAGGCTGTCCC-3´5´-GACGATAGTCATGCCCCGCG-3´5´-CTTTATGTTTTTGGCGTCTTCCA-3´RVprimer3 is especially useful for identifying positions of nested deletions.Note: All three primers can be used for dsDNA sequencing, but onlyRVprimer4 and GLprimer2 also may be used for ssDNA sequencing.Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USAToll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.comPrinted in USA.Part# TM033Revised 9/07 Page 11
VIII. AppendixVIII.A. Common Structural Elements of the pGL3 Luciferase Reporter VectorsExcept for the inclusion of promoters and enhancers, the four pGL3 LuciferaseReporter Vectors are structurally identical. Each plasmid’s distinguishingfeatures are summarized in Section III. The pGL3 Vectors each contain a highcopy-numberprokaryotic origin of replication for maintenance in E. coli, anampicillin-resistance gene for selection, and a filamentous phage origin ofreplication (f1 ori) for single-stranded DNA (ssDNA) production. Restrictionsites for insertion of DNA fragments are located upstream and downstream ofthe luciferase gene. Two of the upstream sites (XhoI and BglII) yield cohesiveends compatible with the downstream sites (SalI and BamHI, respectively),allowing the interchange of the DNA insert for rapid analysis of positionaleffects.Average Relative Light Units1,4001,2001,0008006004002000Improved Expression Levelwith the pGL3-Control Vector28.5pGL2-ControlVector1,350pGL3-ControlVectorConstruct TransfectedFigure 6. Comparison of luciferase activities expressed in HeLa cells transfectedwith the pGL2-Control and pGL3-Control Reporter Vectors. The expression levelof luc+ is dramatically higher with the pGL3-Control Vectors. In repeatedexperiments with several cell lines, we observed 20- to 100-fold higher luciferaseactivity from cells transfected with pGL3-Control. Luciferase activity was measuredwith a Turner Designs luminometer. (Absolute light values and relative expressionprofiles may vary between different cell types.)Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USAToll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.comPart# TM033Printed in USA.Page 12 Revised 9/07
Page 1 and 2: Technical ManualpGL3 Luciferase Rep
Page 3 and 4: I. DescriptionThe pGL3 Luciferase R
Page 5 and 6: III.B. pGL3-Enhancer VectorThe pGL3
Page 7 and 8: III.D. pGL3-Control VectorThe pGL3-
Page 9 and 10: IV.B. Preparation of pGL3 Vectors a
Page 11: VI.Assay of Luciferase Activity (co
Page 15: VIII.C. The pGL3 Vectors luc+ GeneM
Page 18 and 19: VIII.F. References1. Sambrook, J. e
Page 21: VIII.G. pGL3-Basic Vector Restricti
Page 25 and 26: VIII.I. pGL3-Promoter Vector Restri
Page 27: VIII.J. pGL3-Control Vector Restric
Page 30: pGL4 Luciferase VectorsProduct Size

pGL3 Luciferase Reporter Vectors Technical Manual #TM033

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