rpoD sequencing - (Jun/19/2006 )

Hi,
I am trying to sequence the rpoD gene for my bacterial isolates. I am trying to use degenerate primers for this purpose.
I shall be very much obliged if someone could give me some suggestion on it, if someone has already done this before.
I specifically would like to know the PCR conditions used for this pirpose and a suitable source from where I can get my degenerate primers. I am thinking of using the universal degenerate primer available for this purpose.

Thanks.

rpoD is a pretty common gene. What is you're species? Degenerate primers can be cumbersome, sometimes. I'd find the closest related species or common strain used for your bug and try amplify using that gene/genomic sequence. I typed in rpod into Entrez http://www.ncbi.nlm.nih.gov/entrez/query.f...=search&DB=gene and got about 297 hits. If your species isn't there you can use the sequence of the next closest species to begin your degenerate primer design. I can't recommend any specific resource, but I'm sure other members can help, or the protocols listed on this site or peruse the web.

It is also helpful to align several close species to locate highly conserved regions. Look also for Met or Trp amino acids, which have only a single codon, and thus a unique sequence. Similarly, favor the two codon ones in creating degenerate primers. You'd like to push the degeneracy as far as possible toward the 5' end of the primer.