Catalog # N0317 was discontinued on August 02, 2018 ;
The pCLuc-Basic 2 Vector is a cloning vector for expression of Cypridina Luciferase in mammalian cell. It contains the secreted CLuc reporter gene but lacks promoter elements. It can be used to express CLuc under the control of sequences cloned in the MCS.
CLuc can be assayed in the culture medium of mammalian cells expressing Cypridina luciferase
CLuc does not use the same substrate as other marine luciferases, e.g., Renilla and Gaussia. Therefore, it is possible to assay both CLuc and GLuc independently in cell culture medium from cells expressing both reporters.
Can be used to generate Neomycin-resistant stable cell lines
pCLuc-Basic 2 is a cloning vector for expression in mammalian cells, containing a reporter gene but lacking promoter elements. The reporter gene is the secreted luciferase from the Ostracod Cypridina noctiluca. Cypridina Luciferase (CLuc) is a 62 kDa protein with a native signal peptide at the N-terminus that allows it to be secreted from mammalian cells (1) so that CLuc activity can be detected in the culture medium. The pCLuc-Basic 2 Vector contains a multiple cloning site (MCS) upstream of the CLuc coding sequence. A neomycin resistance gene under the control of the SV40 promoter allows selection for stable integration of the plasmid into the mammalian cell genome using G418.
Recommended sequencing primers for pCLuc-Basic 2:
Upstream of MCS:
pBasic Forward Sequencing Primer (23-mer) (not available from NEB)
5´-GGGGTTCCGCGCACATTTCCCCG-3´(6020–6042)
pBasic Reverse Primer (25-mer) (not available from NEB)
5´-TCAGAAGCCATAGAGCCCACCGCAT-3´(1888–1864)
CLuc 3´ End Forward Primer (23-mer) (not available from NEB)
5´-GAGTTCAAGAAAGAATGCTACAT-3´(1671–1693)
CLuc 5´ End Reverse Primer (24-mer) (not available from NEB)
5´-GTAAGGACAGTCCTGGCAATGAAC-3´(143–120)
Figure 1:
Cypridina Luciferase (CLuc) activity obtained from different CLuc plasmidsHeLa cell supernatants (20 μl) and lysates (5 μl) were assayed with
the BioLux CLuc Assay Kit (NEB #E3309). HeLa cells were seeded in 12-well
plates and transfected with 50 ng of CLuc-expressing plasmid per well. At
24 hr post-transfection, supernatants were collected and cell lysed in 100 μl
per well using Luciferase Cell Lysis Buffer (NEB #B3321). The CLuc activity
was measured in a Mithras LB940 (Berthold Technologies) luminometer set
to: 50 μl of injection, 2 seconds of delay and 2 seconds of integration.Figure 2: pCLuc-Basic 2 multiple cloning site (MCS) The Cypridina Luciferase sequence is shown with a blue background. Only unique restriction sites are shown.
DNASU and Addgene are central repositories for plasmid clones and collections that may also be helpful.
Multiple samples can be obtained from the same transfected cells (i.e., before and after experimental treatments or at multiple time points).
90–95% of CLuc activity is found in the cell culture medium, with the remaining 5-10% detectable in cell lysates (Figure 1). This allows flexibility when assaying CLuc along with other co-transfected reporters.
The activity of CLuc is high and the CLuc assay is sensitive enough to detect very small amounts of CLuc enzyme activity.
CLuc does not use the same substrate as other marine luciferases (e.g. Renilla and Gaussia). Therefore, it is possible to assay both CLuc and GLuc independently in cell culture medium from cells expressing both reporters.
The pCLuc-Basic 2 Vector can be transfected into cells using any standard transfection protocol and stable cell lines can be established using Neomycin selection.
Application Features
The pCLuc-Basic 2 Vector can be used to test promoters by cloning promoter element of interest into the MCS upstream of the CLuc reporter gene. For constitutive expression of CLuc, vectors containing promoters are available (See Related Products).
CLuc can be used as a stand alone reporter or in conjunction with other compatible reporters such as Gaussia Luciferase (GLuc) (2). CLuc and GLuc are ideally suited for co-expression as both are secreted and highly active enzymes providing ease of use and sensitivity (2).
The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at [email protected] or fill out the Technical Support Form for appropriate document.)
Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.
The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at [email protected] or fill out the Technical Support Form for appropriate document.)
This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).
New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.
While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.
For more information about commercial rights, please contact NEB's Global Business Development team at [email protected].
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
Licenses
Licensed under certain patents and patent applications from the National Institute of Advanced Industrial Science and Technology ("AIST") for Research and Development Purposes.
For use of the Biolux Cypridina Luciferase Assay Kit, or associated assay reagents, in human diagnosis and measurement in relation to human health, contact [email protected]
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