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Promoter Cloning


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#1 Marvilla

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Posted 24 March 2005 - 04:54 PM

I am trying to clone a promoter controlling the expression of a reporter gene. The promoter has never been cloned before so there aren't plasmids aviable to perform a subcloning. I planned to amplify the promoter from genomic DNA. In order to know the secuence of the promoter I search my gene in the web site of the ensembl project (ensembl.org) and I designed degenerated primers (only 1 mismatch) to amplify 1200 pb from the first exon of the gene to the upstream sequence. I finaly got the amplificate of the exact size I expected, and I cut it with restriction enzimes to be sure that it was the promoter. However the cuts didn't appear where they were supposed to be. So I would like to ask:
1) Is it reliable the sequence of the first draft of the human genome project to design primers for a task like this?
2) Has anybody tried a similar cloning strategy?
3) Is it possible that even though I blasted the primers they are ampliflying other thing of the same molecular weigh?
4) The genomic DNA it was from a southamerican man. How common are the polimorfisms in the promoters to think that they are reponsible for the wrong cutting pattern?
Thanks :o

#2 pcrman

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Posted 24 March 2005 - 08:26 PM

1) Is it reliable the sequence of the first draft of the human genome project to design primers for a task like this?
The human genome sequences are no longer draft but completed.

2) Has anybody tried a similar cloning strategy?
I guess yes, because the sequence is known, you don't need to do a genome walk which is harder. It is simply a PCR cloning.

3) Is it possible that even though I blasted the primers they are ampliflying other thing of the same molecular weigh?
Yes, that's likely. Try UCSC ePCR to see if your primers match other region with a amplicon of the same size.

4) The genomic DNA it was from a southamerican man. How common are the polimorfisms in the promoters to think that they are reponsible for the wrong cutting pattern?
It is possible that the restriction site happens to be a polymorphic site, try another enzyme or do sequencing. Make sure your enzyme works.




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