serching for genes - (Jun/10/2008 )
Dear colleges, I am doing a PHD in plant science and my objective at this moment is find a gene in my plants. Unfortunatelly its genome is no complete so I do not have to much information about genes.... I am using arabidopsis and rice as a modes as there is similar genes in these two plant than this one that I have to find.
I designes primers using the rice sequenece of my gene but I am no getting any results, all my PCR are derty, I mean there are no clera bands and I do not know what to do.
Could anyone help me??
You need to look at the similar genes in all of the organisms sequenced, and try to identify highly conserved residues. The conserved domain database will be helpful. Look especially for uniquely coded amino acids such as Met and Trp. Design degenerate primers in these regions targeting the highly conserved parts of the gene. Ideally, the Met/Trp known bases would be coded by bases at the 3' end of a degenerate primer. Once you have a small section of DNA amplified and sequenced, you can use inverse PCR to expand it.