I'm using Amoa_2R and Amoa_1F primers. After the PCR my gel showed multiple bands for the soil sample that I'm working with. I was expecting only a 481 bp. I tried different annealing temperatures but the result is always the same. Do you have any suggestions or is it normal to have multiple bands with this primers?
Dear Vivian,
I don't know well these particular oligonucleotides you use as primers.
But ammonia-oxidizing bacteriae and archeae are commonly found together in soil samples and their ammonia-oxidizing enzymes exhibit a high varaibility. I attached a recent paper on that topic that may be of some use to you.
Good luck with your experiments,
Pr Philippe Urban
Hi Vivian,
You can go through the following paper for the primer pair which were used for the qPCR of amoA gene.
Singh et al. (2015), Nontarget effects of chemical pesticides and biological pesticide on rhizospheric microbial community structure and function in Vigna radiata. Environ Sci Pollut Res.
I used the primers mentioned in the paper for qPCR and i got a single band of around 481bp.
Good Luck
Regards,
Sunil Singh
Hello Vivian,
i used the primer set during my phD and I can remember that I have played with the annealing temperature to have distingt band. In the end I used 58°C and 35 cycle. Do you use a positiv and a negative control? Like a AOB and AOA?
Regards
Tina
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