I’m currently working on the synthesis of aptamers through SELEX and the technique I’m using for converting dsDNA to ssDNA is asymmetric PCR, however when performing PAGE Denaturing electrophoresis, I cannot see any product bands, just the primer, why is that? for the asymmetric PCR I use only FAM reverse primer 1uM.
In asymmetric pcr you only double the original amount of template dna so you need to run a lot of cycles to make visible amounts of amplimer especially as short ssDNA will not take up much dye so will be hard to see. Other methods like pcr then nucleasing the other strand away may be more productive
If you're performing asymmetric PCR with only one primer (in your case, the FAM-labeled reverse primer), and you're not seeing any product bands on a denaturing polyacrylamide gel electrophoresis (PAGE), there could be several potential reasons for this:
By carefully reviewing these factors and optimizing your experimental conditions, you should be able to troubleshoot the issue and visualize your PCR products on the denaturing gel.
Don't denature the nucleic acids. You can also try running it through an agarose gel instead of polyacrylamide.
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