What is nested PCR?
What is nested PCR?
Nested PCR is a method that involves re-amplification to improve PCR results. Nested PCR involves designing a new forward-nested (FN) or reverse-nested (RN) primer that is internal to the original primer and can pair with the original partner primer. A very small amount of the primary PCR product is used as a template for PCR with nested primers. Nested PCR frequently leads to improved yield of the desired PCR product by: -Eliminating extra bands that may have been present in the initial PCR -Producing a robust band that may have been weak or invisible in the initial PCR It is important to note that only a very small amount of the primary product should be used in nested PCR because this template has very low sequence complexity. To start, the primary PCR product can be diluted 1:100, and 1 µl can be used as the template for nested PCR. Also, you may need to reduce the number of cycles to 25–30. The optimal conditions for nested PCR should be determined empirically.