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1、Te chnica l Ma nua l pGL3 Luciferase Reporter Vectors INSTRUCTIONS FOR USE OF PRODUCTS E1741, E1751, E1761 AND E1771. PRINTED IN USA. Revised 12/08Part# TM033 A F 9 T M0 3 3 1 2 0 8 T M0 3 3 tm033.1208.qxp 12/31/2008 9:15 AM Page a Promega Corporation2800 Woods Hollow Road Madison, WI 53711-5399 USA
2、 Toll Free in USA 800-356-9526 Phone 608-274-4330 Fax 608-277-2516 Printed in USA.Part# TM033 Revised 12/08Page 1 1. Description2 2. Product Components and Storage Conditions2 3. pGL3 Vector Maps and Sequence Reference Points2 A. pGL3-Basic Vector3 B. pGL3-Enhancer Vector4 C. pGL3-Promoter Vector 5
3、 D. pGL3-Control Vector .6 4. Cloning Methods.7 A. Cloning Strategies.7 B. Preparation of pGL3 Vectors and Insert DNA for Cloning.8 C. Transformation Protocols for pGL3 Vectors8 D. Isolation of Plasmid DNA.8 5. Transfection of Mammalian Cells9 6. Assay of Luciferase Activity9 7. Sequencing of Lucife
4、rase Reporter Vectors.11 8. Appendix.12 A. Common Structural Elements of the pGL3 Luciferase Reporter Vectors 12 B. Advantages of the pGL3 Vectors.13 C. The pGL3 Vectors luc+ Gene14 D. Mapping Genetic Elements Located Within DNA Fragments16 E. Composition of Buffers and Solutions16 F. References17 G
5、. pGL3-Basic Vector Restriction Sites.18 H. pGL3-Enhancer Vector Restriction Sites.20 I.pGL3-Promoter Vector Restriction Sites.23 J.pGL3-Control Vector Restriction Sites26 K. Related Products.28 pGL3 Luciferase Reporter Vectors All technical literature is available on the Internet at: Please visit
6、the web site to verify that you are using the most current version of this Technical Bulletin. Please contact Promega Technical Services if you have questions on use of this system. E-mail: tm033.1208.qxp 12/31/2008 9:15 AM Page 1 1.Description The pGL3 Luciferase Reporter Vectors(ac)provide a basi
7、s for the quantitative analysis of factors that potentially regulate mammalian gene expression. These factors may be cis-acting, such as promoters and enhancers, or trans-acting, such as various DNA-binding factors. The backbone of the pGL3 Luciferase Reporter Vectors is designed for increased expre
8、ssion, and contains a modified coding region for firefly (Photinus pyralis) luciferase that has been optimized for monitoring transcriptional activity in transfected eukaryotic cells. The assay of this genetic reporter is rapid, sensitive and quantitative. In addition, these Luciferase Reporter Vect
9、ors contain numerous features aiding in the structural characterization of the putative regulatory sequences under investigation. 2.Product Components and Storage Conditions Product SizeCat.# pGL3-Control Vector20gE1741 pGL3-Basic Vector20gE1751 pGL3-Promoter Vector20gE1761 pGL3-Enhancer Vector20gE1
10、771 Information on related products, including the Luciferase Assay System, is provided in Sections 47 and 8.K. Storage Conditions: Store the pGL3 Luciferase Reporter Vectors at 20C. 3.pGL3 Vector Maps and Sequence Reference Points The listings of restriction sites for the pGL3 Luciferase Reporter V
11、ectors are provided in Section VIII.GJ. Note: The specific transcriptional characteristics of the pGL3 Vectors will vary for different cell types. This may be particularly true for COS cells, which contain the SV40 large T antigen. The SV40 large T antigen promotes replication from the SV40 origin,
12、which is found in the promoter of the pGL3-Promoter and pGL3-Control Vectors. The combination of large T antigen and SV40 origin will result in a higher copy number of these vectors in COS cells, which in turn may result in increased expression of the reporter gene compared to other cell and vector
13、combinations. Promega Corporation2800 Woods Hollow Road Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526 Phone 608-274-4330 Fax 608-277-2516 Part# TM033Printed in USA. Page 2Revised 9/07 tm033.1208.qxp 12/31/2008 9:15 AM Page 2 3.A. pGL3-Basic Vector The pGL3-Basic Vector lacks eukaryotic p
14、romoter and enhancer sequences, allowing maximum flexibility in cloning putative regulatory sequences. Expression of luciferase activity in cells transfected with this plasmid depends on insertion and proper orientation of a functional promoter upstream from luc+. Potential enhancer elements can als
15、o be inserted upstream of the promoter or in the BamHI or SalI sites downstream of the luc+ gene. Figure 1. pGL3-Basic Vector circle map. Additional description: luc+, cDNA encoding the modified firefly luciferase; Ampr, gene conferring ampicillin resistance in E. coli; f1 ori, origin of replication
16、 derived from filamentous phage; ori, origin of replication in E. coli. Arrows within luc+ and the Amprgene indicate the direction of transcription; the arrow in the f1 ori indicates the direction of ssDNA strand synthesis. pGL3-Basic Vector Sequence Reference Points: Promoter(none) Enhancer(none) Multiple cloning region158 Luciferase gene (luc+)881740 GLprimer2 binding site89111 SV40 late poly(A)
