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64 4 Gene Cloning, Expression, and Silencing 4.1.2 pPK-CMV Reporter Vectors 4 Gene Cloning, Expression, and Silencing Applications Transient, high-level reporter gene/ target gene expression in mammalian cells and tissues Monitoring transfection efficiency and gene expression Features High expression of five popular reporter genes Suited for in vitro and in vivo gene expression Convenient multiple cloning site for optimized cloning and expression of target genes Suited for a wide variety of cell types Small vector size ensures high transfection efficiency and high plasmid yield in E. coli The pPK-CMV Reporter Vectors have been designed for high level, transient expression of reporter or target genes. They contain an optimized CMV promoter followed by intron A from the CMV immediate-early gene (IE). This ensures extremely high transient expression in a wide variety of mammalian cells and tissues. In addition, the vectors contain an optimized plasmid backbone that has been extensively altered to achieve optimal plasmid production in E. coli. The vectors are available without a reporter gene to clone and express a gene of interest (blank vector) as well as with the following reporter genes: β-galactosidase (lacZ), green fluorescent protein (GFP), luciferase (Luc), chloramphenicol acetyltransferase (CAT), and secreted alkaline phosphatase (SEAP). pPK-CMV Reporter Vector 4.2 kb Fig. 2: The pPK-CMV Reporter Vector allows strong transient expression of target genes as well as different reporter genes in many cell types and tissues. Product Size Catalog Number Reporter MCS pUC ori Acc I Sal I Eco72 I Bcl I EcoR V Not I Bbe I Ehe I Kas I Nar I BamH I CMV β-Gal GFP Luciferase CAT SEAP pPK-CMV-R1 Reporter Vector (β-Gal) 25 µg PK-MB-P010200 pPK-CMV-R2 Reporter Vector (CAT) 25 µg PK-MB-P020200 pPK-CMV-R3 Reporter Vector (Luc) 25 µg PK-MB-P030200 pPK-CMV-R4 Reporter Vector (GFP) 25 µg PK-MB-P040200 pPK-CMV-R5 Reporter Vector (SEAP) 25 µg PK-MB-P050200 pPK-CMV-R0 Reporter Vector (Blank) 25 µg PK-MB-P000200 HSV pA SV40pA Kan AmpP SV40p For prices please see our online shop at www.promokine.info Please see chapter 1 for luciferase, beta-Gal, and GFP reporter assays.
4 Gene Cloning, Expression, and Silencing 65 4.1.3 pPK-CMV Fusion Vectors Applications efficient selection in both E. coli (using pPK-CMV-F4 (N-Luc) has a Luciferace kanamycin) and mammalian cells (using coding sequence without a stop codon Constitutive high-level GFP or neomycin). inframe in the MCS. The gene of inter- Luciferase fusion protein expression An SV40 polyadenylation signal provides est can therefore be expressed with Lu- Creating GFP or Luciferase efficient transcription termination and ciferase at the N-terminus. expressing stable cell lines polyadenylation - thus ensuring stable mRNA. A pUC origin of replication al- Features Optimized CMV promoter provides high level constitutive expression Suited for a wide variety of cell types lows for high copy numbers in E. coli. The vectors can be used for either transient transfection or for generating stable cell lines expressing only the reporter gene or the fusion target-reporter gene pPK-CMV-F1 (C-GFP, 4965 bp) pPK-CMV-F2 (N-GFP, 4962 bp) Pst I (2) Bgl II Xho I Hind III EcoR I (2) Pst I (2) Bgl II Xho I Hind III EcoR I (2) GFP GFP EcoR I (2) Pst I (2) Kpn I Apa I Sma I Xma I BamH I Not I Xba I Stop EcoR I (2) Pst I (2) Kpn I Apa I Sma I Xma I BamH I Not I Xba I Small vector size ensures high construct. transfec tion efficiency Two multiple cloning sites upstream or downstream of the reporter gene The pPK-CMV Fusion Vectors have been Four versions of the vector are available for convenient and efficient gene cloning and expression of the target gene fused with a reporter gene: pPK-CMV-F1 (C-GFP) has a GFP cod- pPK-CMV-F3 (C-Luc, 5898 bp) pPK-CMV-F4 (N-Luc, 5895 bp) Pst I (2) Bgl II Xho I Hind III EcoR I (2) Pst I (2) Bgl II Xho I Hind III EcoR I (2) LUC LUC EcoR I (2) Pst I (2) Kpn I Apa I Sma I Xma I BamH I Not I Xba I Stop EcoR I (2) Pst I (2) Kpn I Apa I Sma I Xma I BamH I Not I Xba I designed for high-level expression of ing sequence with a stop codon inframe GFP or Luciferase fusion proteins and for creating GFP or Luciferase express- in the MCS. The gene of interest can therefore be expressed with GFP at the + Intron pT7 SV40pA ing stable cell lines. They contain an C-terminus. optimized CMV promoter and an intron sequence, which ensures extremely high constitutive expression in a wide variety of mammalian cell types. Due to two multiple cloning sites on either side of the reporter gene, N- or C-terminal fusion proteins can easily pPK-CMV-F2 (N-GFP) has a GFP coding sequence without a stop codon inframe in the MCS. The gene of interest can therefore be expressed with GFP at the N-terminus. pPK-CMV-F3 (C-Luc) has a Luciferase coding sequence with a stop codon in- pUC ori pPK-CMV Fusion Vector HSV pA Kan/Neo AmpP SV40p 4 Gene Cloning, Expression, and Silencing be generated. In addition, each vector frame in the MCS. The gene of interest Fig. 3: The pPK-CMV Fusion Vectors are contains a combined kanamycin/neo- can therefore be expressed with Lu- extremely versatile, and their compact mycin resistance gene, which allows for ciferase at the C-terminus. size insures high transfection efficiency. Product Size Catalog Number pPK-CMV-F1 Fusion Vector (C-GFP) 20 µg PK-MB-P003400 pPK-CMV-F3 Fusion Vector (C-Luc) 20 µg PK-MB-P003500 pPK-CMV-F2 Fusion Vector (N-GFP) 20 µg PK-MB-P013400 pPK-CMV-F4 Fusion Vector (N-Luc) 20 µg PK-MB-P013500 For prices please see our online shop at www.promokine.info Please see chapter 1 for luciferase, beta-Gal, and GFP reporter assays.
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Cell Biology Products 2011 | 2012
Page 3 and 4:
Overview 1 Overview 1 Cell Analysis
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Contents 3 4 5 6 7 8 Gene Cloning,
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6 Contents Chapter 1 1 Cell Analysi
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8 1 Cell Analysis Bioluminescent Ce
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10 1 Cell Analysis 1.3 Cytotoxicity
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12 1 Cell Analysis Product Size Cat
Page 16 and 17: 14 1 Cell Analysis Product Product
Page 26 and 27: 24 1 Cell Analysis 1.9 Cell Stress
Page 28 and 29: 26 1 Cell Analysis 1.10 Cell Fracti
Page 30 and 31: 28 1 Cell Analysis 1.11 Nucleic Aci
Page 33 and 34: Apoptosis 2 Apoptosis, or programme
Page 35 and 36: 2 Apoptosis 33 2.1 DNA Fragmentatio
Page 37 and 38: 2 Apoptosis 35 2.4 Glutathione Dete
Page 39 and 40: 2 Apoptosis 37 Product Size Catalog
Page 41 and 42: 2 Apoptosis 39 Caspase Screening Ki
Page 43 and 44: 2 Apoptosis 41 Product Size Catalog
Page 45 and 46: 2 Apoptosis 43 2.8 Kinase Detection
Page 47 and 48: 2 Apoptosis 45 2.10 Apoptotic Cell
Page 49 and 50: Fluorescent Labeling 3 The availabi
Page 51 and 52: 3 Fluorescent Labeling 49 3.1 Promo
Page 53 and 54: 3 Fluorescent Labeling 51 Excitatio
Page 55 and 56: 3 Fluorescent Labeling 53 3.2.2 Pro
Page 57 and 58: 3 Fluorescent Labeling 55 3.3.3 Pro
Page 59 and 60: 3 Fluorescent Labeling 57 3.4 Phyco
Page 61 and 62: 3 Fluorescent Labeling 59 3.5 Phyco
Page 63 and 64: Gene Cloning, Expression, 4 and Sil
Page 65: 4 Gene Cloning, Expression, and Sil
Page 69 and 70: 4 Gene Cloning, Expression, and Sil
Page 71 and 72: 4 Gene Cloning, Expression, and Sil
Page 73: 4 Gene Cloning, Expression, and Sil
Page 76 and 77: 74 Contents Chapter 5 5 Cell Transf
Page 78 and 79: 76 5 Cell Transfection Fig. 4: Comp
Page 80 and 81: 78 5 Cell Transfection n Some cell
Page 82 and 83: 80 5 Cell Transfection Phase contra
Page 84 and 85: 82 5 Cell Transfection Magnet Plate
Page 87 and 88: Antibodies and ELISAs 6 Antibodies
Page 89 and 90: 6 Antibodies and ELISAs 87 6.1 Mono
Page 91 and 92: 6 Antibodies and ELISAs 89 n Kinase
Page 93 and 94: 6 Antibodies and ELISAs 91 6.2 Labe
Page 95 and 96: 6 Antibodies and ELISAs 93 6.3 ELIS
Page 97 and 98: 6 Antibodies and ELISAs 95 Product
Page 109 and 110: Cytokines and Growth 7 Factors Cyto
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7 Cytokines and Growth Factors 115
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138 Contents Chapter 8 8 Microbial
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140 8 Microbial Detection and Elimi
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142 8 Microbial Detection and Elimi
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144 Alphabetical Index Alphabetical
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146 Alphabetical Index Description
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160 Alphabetical International Inde
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162 International Alphabetical Inde
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Notes Notes 165
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Terms and Conditions 167 4. The rig
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Fax Order Form Shipping Address Inv
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