HI...
i have some strain agrobacterium rhizogenes and I want to know variation between my straines i choiced a primer from article and i blast that primer in NCBI ,result blast is : product lenthg is 914bp and i did pcr with that primer but i have other bands except 914bp
my primer is :
F: GCTATGCATGGCACGAC
R: CCCACGGCTTTTCGCAG
my question:is that primer appropriate for finding variation between my straines or no?
and other question , which band is appropriate for sequncing?
i send you my gel of photo
I would try improving your pcr first before cutting and purifying bands as a single clean band can be sequenced better and easier and cheaper. Try increasing the annealing temperature preferably in a gradient cycler to try to achieve a single clean band. Alternatively run a pcr with increasing amounts of dmso from 0 in 1% steps up to 8% dmso and one of these reactions should produce a clean product since dmso increases the specificity of the primers so should get rid of unwanted bands
I would try improving your pcr first before cutting and purifying bands as a single clean band can be sequenced better and easier and cheaper. Try increasing the annealing temperature preferably in a gradient cycler to try to achieve a single clean band. Alternatively run a pcr with increasing amounts of dmso from 0 in 1% steps up to 8% dmso and one of these reactions should produce a clean product since dmso increases the specificity of the primers so should get rid of unwanted bands
I think you need to use better primers and also enhance the PCR situation as Paul explained. Your primers seems to be very short in length and I believe it is causing unspecific bonds.
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