pGL3 Luciferase Reporter Vectors Technical Manual #TM033

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pGL3 Luciferase Reporter VectorsAll technical literature is available on the Internet at: www.promega.com/tbs/Please visit the web site to verify that you are using the most current version of thisTechnical Bulletin. Please contact Promega Technical Services if you have questions on useof this system. E-mail: techserv@promega.comI. Description..........................................................................................................2II. Product Components and Storage Conditions ............................................2III. pGL3 Vector Maps and Sequence Reference Points ..................................2A. pGL3-Basic Vector................................................................................................3B. pGL3-Enhancer Vector ........................................................................................4C. pGL3-Promoter Vector ........................................................................................5D. pGL3-Control Vector ...........................................................................................6IV. Cloning Methods ...............................................................................................7A. Cloning Strategies.................................................................................................7B. Preparation of pGL3 Vectors and Insert DNA for Cloning...........................8C. Transformation Protocols for pGL3 Vectors ....................................................8D. Isolation of Plasmid DNA...................................................................................8V. Transfection of Mammalian Cells..................................................................9VI. Assay of Luciferase Activity............................................................................9VII. Sequencing of Luciferase Reporter Vectors...............................................11VIII. Appendix ...........................................................................................................12A. Common Structural Elements of the pGL3 LuciferaseReporter Vectors ................................................................................................12B. Advantages of the pGL3 Vectors .....................................................................13C. The pGL3 Vectors luc+ Gene ............................................................................14D. Mapping Genetic Elements Located Within DNA Fragments....................16E. Composition of Buffers and Solutions ............................................................16F. References............................................................................................................17G. pGL3-Basic Vector Restriction Sites.................................................................18H. pGL3-Enhancer Vector Restriction Sites.........................................................20I. pGL3-Promoter Vector Restriction Sites.........................................................23J. pGL3-Control Vector Restriction Sites ............................................................26K. Related Products.................................................................................................28Promega 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 1
I. DescriptionThe pGL3 Luciferase Reporter Vectors (a,b) provide a basis for the quantitativeanalysis of factors that potentially regulate mammalian gene expression. Thesefactors may be cis-acting, such as promoters and enhancers, or trans-acting,such as various DNA-binding factors. The backbone of the pGL3 LuciferaseReporter Vectors is designed for increased expression, and contains a modifiedcoding region for firefly (Photinus pyralis) luciferase that has been optimized formonitoring transcriptional activity in transfected eukaryotic cells. The assay ofthis genetic reporter is rapid, sensitive and quantitative. In addition, theseLuciferase Reporter Vectors contain numerous features aiding in the structuralcharacterization of the putative regulatory sequences under investigation.II.Product Components and Storage ConditionsProduct Size Cat.#pGL3-Control Vector 20µg E1741pGL3-Basic Vector 20µg E1751pGL3-Promoter Vector 20µg E1761pGL3-Enhancer Vector 20µg E1771Information on related products, including the Luciferase Assay System, is provided inSections IV–VI and VIII.K.Storage Conditions: Store the pGL3 Luciferase Reporter Vectors at –20°C.III.pGL3 Vector Maps and Sequence Reference PointsThe listings of restriction sites for the pGL3 Luciferase Reporter Vectors areprovided in Section VIII.G–J.Note: The specific transcriptional characteristics of the pGL3 Vectors willvary for different cell types. This may be particularly true for COS cells,which contain the SV40 large T antigen. The SV40 large T antigen promotesreplication from the SV40 origin, which is found in the promoter of thepGL3-Promoter and pGL3-Control Vectors. The combination of large T antigenand SV40 origin will result in a higher copy number of these vectors in COScells, which in turn may result in increased expression of the reporter genecompared to other cell and vector combinations.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 2 Revised 9/07
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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 and 12: VI.Assay of Luciferase Activity (co
Page 13 and 14: VIII. AppendixVIII.A. Common Struct
Page 15: VIII.C. The pGL3 Vectors luc+ GeneM
Page 18 and 19: VIII.F. References1. Sambrook, J. e
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Page 25 and 26: VIII.I. pGL3-Promoter Vector Restri
Page 27: VIII.J. pGL3-Control Vector Restric
Page 30: pGL4 Luciferase VectorsProduct Size

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