Primers: advice? - (Apr/22/2004 )
I am about to start a project that looks at different microbial communities in and out of harmful algal blooms, as well as at different depths in these blooms. We will visualize them with DGGE's, cut out bands, clone them, and sequence. In the past, my lab has used the primers 517-341(GC), but we feel that to get a more comprehensive feel for what these communities are, we should use primers that yield more than a 176 bp product. So, we started using the primers 907-341(GC). I just completed a DGGE today, and there is a significant decrease in the amount of bands that showed up with my new primer set vs the amount of bands I get for my old primer set using the same sample. Since the entire crux of the project is identifying as many bacteria as possible, our new primers do not seem to be efficient enough for our purposes. I know this is because of PCR bias, etc...but I am still interested in using a larger PCR product than our original 176 bp. Any suggestions on what other sets I could use? I guess something that is smaller than 566 bands and larger than 176 bands. Also, nothing too high in the 16S gene, such as 1492 or others. We have found that there is too much variability at those sites. Any help would be appreciated!! Thanks!!!
Sorry, posted wrong.