I designed a primer pairs with the expected size of my product is ~750. I checked with primer-BLAST and it returns only 1 product. But when I do PCR and agarose gel electrophoresis, I see 3 bands of the size ~1200, ~1000, and ~350 and no band of the size ~750. I have tried changing annealing temperature, increasing template concentration and performing touchdown PCR but nothing works. What else could I do?
1. design new primer sets and try again
2. sequence the PCR products you are obtaining to check whether you are at all amplifying the desired gene/genomic fragment
It would be helpful if you could provide more detailed information regarding the type of PCR you are trying to establish: RT-PCR, genomic PCR, species,...?
I was trying to perform PCR with the template as human genomic dna and then I need to purify it for sequencing afterward
Thanks for the clarification of your PCR approach. As suggested, I would design other sets of primers and repeat the PCR; and make sure to use genomic DNA isolated from any normal tissue.
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