How can I clone the promoter region of my gene - (May/30/2002 )
Now I have the genomic library and want to clone out the whole promoter region of my gene of interest. What should I do . After I screen the lamda phage library using my gene cDNA as probe, what should I do to make sure I get the whole region of the promoter?
After you get the positive clones from you library , I think the best way is that you sequence the part of the clone using the lamda arm sequence as primer (the arm far away from the cDNA of your gene which you used as probe) ,then get the result align in the database to make sure whether you have got the whole sequence of your promoter.
First you must be sure is the region really the promoter or might it be that there is an excisting not translated Exon. For this propose I would perform a 5`-RACE or Primer Extension. Next step: If you know the sequence order
primer, make a PCR and clone the fragment. If not the simplest wy is first to sequence the promoter region tnah make a PCR or subcloning.
If you are going to perform reporter assays I would start with a fragment up to 1500kb of the 5`-promoter region, but also construct shorter constucts
Thanks for your help,DJ.
It seems it is not an easy job to get a gene's promoter region even there are publications shows where it is and what the sequence is .