Control Constructs

SwitchGear offers a number of control constructs to optimize experimental conditions and to normalize for non-specific effects: Promoter Control Constructs and 3’UTR Control Constructs

 

 

CONTROL CONSTRUCTS FOR PROMOTER REPORTER ASSAYS

SwitchGear offers a number of control constructs to optimize experimental results. Use the Promoter Construct Protocol for transfection of these vectors. We recommend using at least one of each vector described below for best experimental results.

Empty Promoter VectorEmpty promoter vector: The empty promoter vector contains the luciferase gene without a promoter. This construct serves as a one measure of background signal in the experiment.

Housekeeping gene promoter vectors: Housekeeping control constructs contain promoters for common housekeeping genes driving the luciferase reporter. These constructs serve as positive transfection controls and may also serve as controls for comparing signals between conditions if they are known to be unresponsive to the test condition. See SwitchDB, our online catalog, for details about these vectors.

Random control vectors: Random control constructs contain 1 kb non-conserved, non-genic, and non-repetitive fragments from the human genome cloned upstream of the luciferase reporter. These vectors produce slightly higher signals than the empty vectors and are considered optimal negative or background controls. See SwitchDB, our online catalog, for details about these vectors.

promoter chart Figure 1. Expected data and analysis of housekeeping and random promoter vectors: Random promoter constructs (R1, R2, R3, R4) serve as negative/background controls and establish baseline luciferase activity. The housekeeping promoter controls serve as positive controls and give relative luciferase activity.
Y-axis values shown in log scale. Control vector activity may vary depending on cell line, treatment condition, and protocol changes.

SwitchGear also offers a set of eight technical control constructs for larger-scale studies to normalize for plate-to-plate variation. Studies involving multiple plates per condition should include these 8 controls. These plate controls represent a combination of housekeeping and random sequence controls.

 

 

CONTROL CONSTRUCTS FOR 3’UTR REPORTER ASSAYS

SwitchGear offers a number of control constructs to optimize experimental conditions and to normalize for non-specific events that may cause signal variation. We recommend including at least one control of each type listed below to minimize the possibility of experimental conditions interacting with a control.

Empty 3'UTR Reporter VectorEmpty 3’UTR vector: The empty 3’UTR vector contains only the luciferase gene (Luc2P) and its constitutive promoter with the multiple cloning site removed. This construct may serve as a positive control for the transfection.

Housekeeping gene 3’UTR vectors: Housekeeping control constructs contain the 3’UTRs for common housekeeping genes cloned downstream of the luciferase reporter. See SwitchDB, our online catalog, for details about these vectors.

Random 3’UTR control vectors: Random control constructs contain non-conserved, non-genic, and non-repetitive human genomic fragments.

See SwitchDB, our online catalog, for details about these vectors.

utr chart Figure 1. Example data for SwitchGear 3’UTR control vectors. Here 50ng of each vector was transfected with FuGene6 (Roche) into HT1080 cells. Absolute and relative activity will vary depending on cell line, experimental conditions, and transfection reagents.
Values shown in log scale. Relative activities will differ depending on cell type and conditions.