how to design primers for 16sr RNA - (Mar/27/2012 )
Conserved hypothetical means that the protein is found in many other species. You may want to look for hypothetical proteins that are not conserved.
Dear All, I want to say "thank you" to everybody who helped me in this problem. I am working on it now. I will let you know when I get process! I appreciate your time and kind help!
Hi! I have found some hypothetical proteins that uniquely expressed in my interested bacteria. I designed primers based on a protein sequence, and the PCR result is good.
I have question here: What if the expression level of this protein in the bacteria is different/unstable. In that case, I cannot use it for quantification (qPCR) of the bacteria? As the proteins are hypothetical, there is no study about it and it is hard to say if the protein expression is stable.
Thanks a lot!
phage434 on Thu Mar 29 19:55:06 2012 said:
Well, if you have sequence, then you can choose a gene that occurs rarely in other species. Look for "hypothetical protein" and blast the protein sequence against the protein database (or you can use "Blink" which has pre-computed this result). If the only hits are in your target organism, you have a unique gene which you design primers for. For best results, I'd do this with several genes and do PCR with all of the primer pairs, as a cross-check.
You are looking at genomic DNA, so the expression level is completely irrelevant. If the gene is there, your PCR will pick it up. There will always be one copy per genome (well, perhaps a few during rapid growth).
Do you recommend I try several genes? Thanks a lot!
phage434 on Mon Jun 11 21:44:01 2012 said: