Posted 19 April 2011 - 11:36 PM
I'm totally new to pGL3 vector and transfection experiment. I'm reading the Promega pGL3 vector user manual at the moment, before planning my experiment. I notice that there are totally 4 types of pGL3 vectors: the control vector which has both SV40 promoter and enhancer, the pGL3-promoter vector that only has the SV40 promoter, the pGL3-enhancer vector that only has the SV40 enhancer and the pGL3-basic vector that lacks both promoter and enhancer.
At first, I think to study my putative promoter just need to clone that DNA fragment into the pGL3-basic vector and compared it with the pGL3-control and pGL3-basic vector. However, when I take a look of an assay experiment on the manual, I'm surprised that only the pGL3-control vector has high luciferase activity, the other 3 vectors: pGL3-basic, pGL3-enhancer and pGL3-promoter, all with nearly none luciferase activity.
So, if I just want to study putative promoter, I need to clone it into pGL3-enhancer vector and not the pGL3-basic vector? (It seems that just with the SV40 promoter still have only basal luciferase activity and my putative promoter may not as strong as SV40.)
Thank you for your help.
Posted 20 April 2011 - 05:38 AM
For your experiment in testing a putative promoter, you will want to use the pGL3-Basic vector. There has always been confusion about the use of the enhancer and "promoter" vectors primarily because what most people call the SV40 promoter is actually both the promoter and enhancer. Both are needed for the typically strong expression from this promoter, and the pGL3 enhancer and promoter vectors can be used to analyse promoters that may benefit from the SV40 enhancer or upstream elements that lack a promoter.
Hopefully this has helped clarify your understanding of the different products and verifies that your original plans were correct. If you have any additional questions or concerns, I recommend getting in contact with Promega Technical Services. We have a global team of scientists very capable of helping you in experimental design and analysis.
Posted 20 April 2011 - 06:46 PM
Thank you for your prompt reply. Your explanation is very useful and clear to me.
I have one more question..... For most of the papers I read using the pGL3 vector, they usually got the results that the luciferase activites of their putative promoter were lower than that of the pGL3 control. Is this expected for any such kind of experiment? If yes, why?
Posted 22 July 2013 - 06:01 PM
we usually use pGL3-control vector as a positive control when performing transfection.
In general, we should use 3 kinds of vectors:
1. pGL3-basic vector containing your insert.
2. pGL3-basic vector without insert as a negative control. You would compare the promoter activity of your construct (pGL3-basic + insert) with this pGL3-basic vector.
3. pGL3-control vector as a positive control to confirm the transfection efficiency.