Cloning a promoter sequence from genomic dna - (Feb/14/2007 )
I need to isolate the promoter of a couple of genes and place in a vector in front of a luciferase reporter. I was just wondering if anyone had any advice on how to design the primers to amplify approx 2kb of the genomic sequence (i.e. do I go up to the exon? The ATG if it's partway through exon 1? etc).
I assume you already have this seqeunce. If not you can obtain it from genomic databases. Design your primers to have a Tm of around 60 degrees. You can add restriction sites directly to the ends of your primer to clone it into the expression vector. It is usually a good idea to include 5' untranslated sequences, i.e. right up to the ATG.
Thanks, that's great!
Yeah I am wondering about the same thing. If I want to test a promoter sequence with luciferase gene (in PGL3), do I also add the 5` untranslated region of the promoter?? what happens to this mRNA transcribed then from my promoter sequence before the coding area of the luciferase? will it be just a fusion mRNA but the translation starts anyway from the luciferase?