It is an aquatic monocot system. Can u pinpoint the techniques that I can use.

I have only around 190bp genomic DNA sequence from this species which I can exploit for primer designing but problem is it is GC rich. Moreover I have tried 3' RACE twice using different tissues but couldnot succeed. When I chk with primers which amplified 190bp fragment in the cDNA pool, there was no amplification, indicating no transcript of my interest in the cDNA pool. That is y, I am not confident with RACE. Same was the case with 5' RACE.

Hope nw u understand my problem.

Looking forward for ur suggestions.

Regards

Mahalakshmi

Thanx 4 the suggestions, but I have checked the sequence contains no intron in that region and even if it wud b there, it won't b there in the cDNA. cDNA library preparation would be a costlier affair. As suggested by u as well I am fully concentrating on homology based primers and degenerate primers. Can u suggest any software or specific criteria to be considered during degenerate primer designing. I have no expertise in its designing so not confident about it

Thanx a ton

Regards

Mahalakshmi

Hi, I read your problem. I was also having a similar kind of a problem while designing degenerate primers. The first problem was, how to choose the related species for the gene of interest for your species. If you are using homology based primer designing, it is most important that you choose related organisms.But what are the criterias, I am really confusing about. For one of my genes, i used 11 primers, but didnot get any results. I choose related organisms depending on classification.But later I realised that there are other several things like, thermostability, pH range etc of a particular gene for any organism to get the changed sequences fromother related organisms.So these are a huge problems for primer designing. If you get any good ways, please let me know. And then RACE is a completely dependent processon primers. even genome walker technique is also for an unknown gene.

Regards

prabuddha Sarkar

Hey!!! thanx Wilco n prabuddha for ur suggestions....I will surely try it n let u knw....

Thanx

Regards

Mahalakshmi