I will do the reporter gene by luciferase assay to study the promoter activity?i will use pgl3 basic vector and prl-tk,, the cell line MCF-7and MCF-7 , I will use lipofectamine 2000 and optimum media
can you give the steps of method ?
do I transfer pgl3 basic without promoter as control in both cell lines?
how to analysis the data ?
Yes always include as much control as you can, and empty vector is always a good control to add.
LifeTechnologies which manufactures Lipofectamine provides a protocol for the transfection of MCF-7 with plasmid DNA, though they use Lipofectamine LTX instead of Lipofectamine 2000. What will change is you need to test the volume of plasmid DNA and Lipofectamine 2000 to achieve a good transfection without high cell death, though I'm pretty sure you can use the same volumes as in the protocol as there is probably not much difference in both reagents. I suggest to test the protocol with the exact same parameters with Lipofectamine 2000 with a few wells and see if you achieve a satisfactory bioluminscence and good cell viability. Good luck !
http://www.lifetechnologies.com/fr/fr/home/references/protocols/cell-culture/transfection-protocol/transfecting-plasmid-dna-into-mcf-7-cells-using-lipofectamine-ltx-reagent.html
i do not use any protein, only i want to study the activity of full length of promoter activity of STK11 gene and relate to the gene expression which i did it before ?
i want to see the promoter activity in normal cell line mcf-10 and breast cancer line mcf-7 and compared between them, ,,,
what i should transfer in each well of reaction ? how to calculate the promoter activity ?
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