cloning of promoter - (Aug/08/2006 )
Hi dear researchers,
I have a very general question: If I want to clone promoter sequences of a given gene in front of a gene (like GFP or any other ), what do I have to take into consideration? It doesn't need to be in frame? Which sequences does it need to contain? And what if enhancer elements located somewhere else (like maybe 3' of the gene) are necessary for the expression? Where do I clone them?
Look for restriction sites upstream of the gene. Get suitable restriction sites on both the 5' and 3' of the promoter and vector u r planning to clone into. the promoter doesnt have to b inframe.
Try to leave some space (more than 60 bps, i ahad trouble getting expression when space was less than 50 bps) )between the promoter and the transgene of choice.
Did you check if your promoter is reported already (looking for in papers or the sequence in NCBI)??, if it is… you can try what they reported. Do you know your size promoter?
I suppose you have a vector with GFP (or any other you want to express), add suitable restriction enzymes sites to the primers you plan to use to amplify your promoter.