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cloning antisense promoter in pGL3Basic
2 replies to this topic
Posted 22 October 2012 - 04:37 AM
Hi, I want to clone a fragment of a promoter in pGL3Basic to analize luciferase activity. I use the XhoI and BglII to clone the fragment. When I checked the sequences after cloning I realized that the promoter have been cloned antisense, BglII-XhoI. I have no explanation for that, but my doubt is if this orientation could affect to the luciferase activity. Please, any help will be greatly appreciated.
Posted 22 October 2012 - 09:10 AM
The orientation of promoters matters. Some promoters do have bidirectional transcriptional activity. But you are looking for just one way of transcription, you got to get the orientation right for luciferase reporter assay.