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Can anybody help me in checking the specificity of my primers. I have chosen ITS, rbcL, matK, and trnH-psbA genes for barcoding of medicinal plants. Primers are taken from literature. I have obtained results of PCR with ITS as well as with rbcL, and trnH-psbA but none of the band exceeded 700bp. I am not sure whether my required region is amplified or not. How can I verify the specificity of primers?
Waiting for the valueable suggestions.
i suugest that you change the extintion time in thermal cycler parameters to read more than 700bp
It is fine if rbcL and ITS are between 500 and 700 bp, but matk should be about 1000bp or larger. I would go for sequencing and compare the sequence with reference data online.