I made detection primers to detect Antimicrobial Resistance Genes in soil that I used in conventional PCR.
But, my primers have different results in different soil samples (I extract DNA in the soil). In one sample the primers have single band, but in others it has many bands.
I tried to increase temperature, in one sample the primers were specific (single band) but others the band was lost (before increasing temperature there was single band in that sample. Are my primers not specific? And how to solve the problem?
Thank you ever so much
gene sequences are usually quite similar between species as they are conserved by being passed on betwen the generations but mutations wi;; sti;; happen, I think that your soil samples have different organisms with different sequences between the primers possibly relecting intron sequence variation and multiple bans are to be expected, Sequencing and Blast analysis may indicate where the many bands are coming from
as Poul said you may have selected a primer pair that isnt as specific as you need. But although you get more than one band as a result you must pay attention just to the specific length of interest. you dont care the other bands at other band size, you know that the primers were designed to amplify an specifc length of pb but they may matched whith other DNA sites of your organism or with other similar DNA sites of other organism.
You need a positive control of your organism and run a sample of it in each pcr reaction, and compare your sample results with the Ladder length marker and your positive control.
Other strategy to better identify your interest sequence is to designed a second pcr with inner primers (nested pcr) and run it whit your suspected positive samples. The best way to know what you have obtained is to sequence the band and blast it as Poul says above.
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