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Asymmetric PCR is a modification of the polymerase chain reaction (PCR) that uses an unequal ratio of primers to amplify one strand of the target DNA more efficiently than the other. In traditional PCR, the ratio of forward and reverse primers is usually 1:1, and both strands of the target DNA are amplified equally. However, in asymmetric PCR, the ratio of forward and reverse primers is usually much higher, such as 1:10 or even 1:100, resulting in preferential amplification of one strand of the target DNA. Asymmetric PCR is often used to generate single-stranded DNA (ssDNA) or RNA for various applications, such as sequencing, cloning, and probe synthesis. #gelelectrophoresis #pcr #dna Question: Why my ssDNA band showed higher than dsDNA on agarose gel? I'm doing asymmetric PCR to amplify ssDNA, using F:R=1:10 ratio of primers. I already optimized the PCR cycle, annealing temperature of my PCR conditions, but when I detected my product with 1% agarose gel, ssDNA band detected higher than the dsDNA band. As i know, ssDNA has to be lower than dsDNA because they migrate faster. Stranger thing is dsDNA band from the asymmetric PCR product showed higher than the normal (F:R=1:1) PCR product band. My target product's size is 185bp. Does anyone tell me what is wrong? I'm using 1% agarose gel, 70V 30-40min (same as DNA ver. agarose gel - I confirmed that electrophoresis gel condition for DNA did not affect to ssDNA detection)
