Questions about proper technical approach - (Jan/03/2013 )
Helow,
I think I found an active promoter region on the human genome, and I guess a certain gene is expressed by the promoter.
Problem is I don't know how to design a series of experiment in order to the promoter that I found is leading to a gene expression.
Could you give me a clue?
Thanks a lot of your helps in advance.
Maybe you have to introduce this region upstream to a mammalian expression vector. Cut and Paste the new Promotor. Little bit tricky would be to use recombination and deleting the region.
Thank you, Pangea, for the reply.
But, now that I see my question, it's kind of wrong, and even my english is terrible. I'm really sorry about that.
My situation is like this.
I found an active promoter on the genome, and I confirm its activity by reporter assay.
Now I'm thinking that there may be a gene which is located down stream of the promoter that I found, and the gene is under the control of the promoter.
To prove my idea about a gene that may be located down stream of the promoter, what should I do? What is the best approach to identify the gene that might be there?
I have no idea what to do regarding this.
Could you give me a clue?
Thanks a lot of your helps in advance.
Maybe you can check first whether it is a gene or not by using biotool like finding ORF, start and stop codons etc. Than you should clone this region if possible with this promotor.
Hi Mindologist,
I'll go straight to your first question. The first thing that came to my mind is to do what's called the genome walking. Bear in mind that I have not done this (or anything similar) before so someone here might have better alternatives.
Here are some stuffs that could be useful:
www.clontech.com/xxclt_ibcGetAttachment.jsp?cItemId=17326
http://www.protocol-...osts/37995.html
www.ucl.ac.uk/~ucbhjow/b200/Cloning_genomic_DNA.doc
http://www.biotechni...ques-44287.html
https://www.ncbi.nlm...cles/PMC306810/
Julio sounds good. Something new again. Thanks for sharing you knowledge.
Thanks a lot, Pangea and Julio, for the good information and knowledge.
One question for Pangea. Since I don't know anything about bioinformatic tools, do you have your favorite one? or could you recommend one of the tools?
Thanks again.