how to clone the promoter of a gene - (May/06/2008 )
I am trying to find out the binding of some transcription factors to a target gene. I plan to do EMSA to get the TFs binding to the promoter and use Mass Spec to identify the TFs in that region. But I am totally new in this area and don't know how to clone the promoter. It is around 350bps long. Can I use PCR to clone the promoter region (sequence is known)?
Any suggestion would be appreciated.
Thank you very much!
PCR should work. It may be easier to assemble it with synthetic oligos.
For gel shift assay, you don't need to use such long DNA probe that is OK for footprinting assay. You can just synthesize oligos of around 20 nt and make a double-stranded oligos by annealing two complementary ones. The oligos should be the sequence of the putative binding site of a transcription factor.
I have used some transcription binding prediction software to predict the putative transcription binding sites. So you mean I can just use these sites for synthesizing the oligos? Thank you very much!