Difluorokinase assay

Summary

Luciferase plasmids were constructed and then transfected into cells to detect alterations in reporter gene expression by comparing overexpression or interference with miRNAs.

Principle

The original transcriptional regulator of the target gene was constructed into an expression vector with firefly luciferase (luciferase), which was constructed into a reporter gene plasmid, so that this sequence regulates the transcriptional expression of luciferase. The reporter gene plasmid is then transfected with cells, which are lysed after giving them different treatments, and the substrate luciferin is added, which is catalyzed by luciferase to emit fluorescence (the strongest wavelength is around 560 nm). The high or low fluorescence value obtained can be detected to determine the effect of different treatment groups on this transcriptional regulator. In order to avoid errors caused by differences in the efficiency of plasmids when transfecting cells, a reporter gene plasmid for Renilla luciferase is usually transferred as an internal reference (strongest wavelength around 465 nm), i.e., a dual fluorescence reporter system.

Appliance

Gene regulation, non-coding RNA targeted interactions and other research areas

Operation method

Difluorokinase assay

Materials and Instruments

【Materials】Specialized fragments; gene vector pGL3-basic; phRL-TK; LAR II; 1X PLB; Stop&Glo; cell lysate

Move

1, Construction of reporter gene plasmid. Insert the target fragment into luciferase-expressing reporter gene vector, such as pGL3-basic.2, Transfection of cells. Co-transfect cells with the reporter gene plasmid and phRL-TK (internal reference), and treat the cells as needed. When co-transfecting, the amount of reporter gene plasmid:internal reference transfection is generally 10:1~50:1 because the internal reference has a strong promoter.Luciferase activity assay：

（1） For initial use, LAR II, the substrate for Firefly luciferase, was prepared. Dissolve LAR II in LAR II buffer and store in portions -80°C away from light.(2) Add 1X PLB and lysed cells for 15 min at room temperature.(3) Prepare Stop&Glo, a substrate for Renilla luciferase, capable of terminating the reaction of LAR II.(4) Determine the fluorescence value. Add 10 ul cell lysate to 40 ul of LAR II, blow and mix, and detect the reading, which is the value of Firefly luciferase. Add 40 ul Stop&Glo and read again, that is the value of Renilla luciferase.(5) Data processing. First calculate the ratio of Firefly luciferase/Renillaluciferase for each tube, and then use the ratio of the control group as unit 1 to get the relative luciferase activity of the different treatment groups, that is, the regulatory activity of gene transcription in that treatment group.

Caveat

1, in order to ensure the stability of luciferase detection reagents can be taken after appropriate partitioning and stored away from light, to avoid repeated freezing and thawing and prolonged exposure to room temperature.

2， in order to achieve the best detection results, the same batch of samples is best to ensure that the same determination time, usually 10 s.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/