Hi,

That is a very long story..

But first you can try to go to NCBI and search for B. subtilis genome published there (select the strain that you are interested). Use that genome to design primers by the online tool Primer3 (or any program that you have, Vector NTI for example).

You have to choose primers that specific for B. subtilis. But primers have to b designed depending on your method of quantification. So please be more specific about your quantification method.

Goodluck.

Duy.

Hi Duy,

I find one article published two primers for Bacillus subtilis with one probe. My question is , if I can used so I shortened the time.

I used standard quantification. I have twob targets, pathogens and antagonist.

Our objective was to quantifiy the effect of antagonist on pathogen and also to be sure that antagonist colonize the root or not (endophyte or not).

I study the mode of action of bacillus

Thanks

Hi,

That's a very interesting topic! It's good that you found an article with primers. But in my experience, those primers would never work in our specific situation for many reasons, maybe different strains and other thing. So you always have to check those primers in NCBI to see if they are match with your target gene and specific for your strain. Then order them and run test PCR (in several strains, and also the antagonist) to make sure it works.

But you still need primers specific for the antagonist too. So You can design and select primers that have similar length (22-25bp), GC content(45-65%), Tm temperature (for Realtime-PCR it should be around 60-65, so the Ta could be around 60, but again it depends on the primers that you already had from the article in case that they work well), and the amplicon should also have those caracteristics similar to those of primers for B. subtilis (size of amplicon for Realtime-PCR should be around 100-150 bp).

Goodluck,

Duy.

THanks Duy,

I inform you if these primers work or not. I try the experiment maybe in two weeks.

Thanks