Dear Leo,

I think it will be a good idea if you digest an amount of your isolated DNA with a restriction enzyme to confirm if the vector has been cut which can be seen a band at ~4.5kb and the rest of your bands should the total size of your construct.

My best regards,

Hello, If you would to be sour successful transformation. I had same problem before. Perform a PCR using your insert DNA primers or universal vector primers. directly from bacterial colony which grown in LB agar with Amp antibiotic, a prokaryotic selection antibiotic for PGL3. For a control do a PCR reaction use human genomic DNA instead the bacterial colony to eliminate DNA contamination.

Do a PCR on your insert (preferrably with one primer from the vector backbone and one from the insert to avoid false positives) and also do a restriction digest.

With the transfection into cell lines always do a control with an empty luciferase vector. This is especially important for pGL2/3 vectors, since the contain cryptic sequences which can be activated in cells. This caused a lot of problems for me, keep it in mind.

Not quite sure at what level of controls you are talking about. I assume you have checked your constructs are cloned properly and have sequenced them. If you are referring to after transfection to make sure of the promoter activity, either a scrambled promoter sequence or non-related promoter may be good - this will show it is your promoter target that is specific. A positive control which usually comes with the kit so show your system is working will help as well.

Leo- most people prefer to run dual luciferase assays where you co-transfect pGL3 and a control luciferase plasmid as an internal control. So I'd suggest you use TK-Renilla or CMV-Renilla in parallel with your pGL3 construct, and measure the ratio of Firefly to Renilla luciferase activity in your assay.

Once you test whether your promoters are active and respond to stimuli of interest, you will want to consider generating mutations in your reporter that would be expected to disrupt the biological response you are measuring. Typically, you would introduce some point mutations in motifs of interest, or delete the sequences from your promoter. While Christian has a good point that empty pGL3 vector is an important negative control, the best negative control is to have a mutant promoter that no longer responds to your signals.

You can try Restriction Digestion using enzyme and then run the gel for getting appropriate band.

I agree with Daniel too. If you have the chance, I would suggest you to try in parallel different controls of transfection efficiency, pTK-Renilla and pCMV-Bgalactosidase to check which one works better in your cells. After measuring both activities you have to calculate the ration luciferase activity / renilla or Bgalactosidase activity values. Of course you have to transfect some wells with the vector pGL3basic without any promoter as a negative control and to calculate he ratio between both activities that will give you the number of times that your promoters induce luciferase gene expression (normalization). Additionally, as Kevin suggested you, I would transfect some cells with a vector expressing GFP but just to check the transfection efficiency of your cells and that the protocol and reagent you are using is the good one. But this proof before performing the big experiment.

Bastian- I haven't seen any intron-containing Renilla constructs, but it wouldn't be too difficult to generate it. There are several promoters that contain intronic sequences as well as synthetic introns that you could insert into your Renilla cDNA. Can send you specific sequences if needed.

Thank you all for suggestions.

I used Renilla vector as dual luciferase assay and it worked well.