Hi guys, As I am going to do the Miseq sequencing. I wanted to run the 1st PCR with 2 cycles, and input the 1st PCR product to the 2nd PCR, which I can run over 20 cycles (I need to test the minimum cycle number of course).
To confirm this strategy works, I used 50ng genomic DNA, run the PCR with 40nM primer each, for 2 cycles (total 25ul volume). I also ran 32 cycles as a positive ct to ensure the PCR works. Later, I input 1ul, 5ul, 12.5ul and 25ul of the PCR product from the 1st PCR to the second PCR, with 500nM primers and 35 cycles. However, I could not get any band from this reaction, except that I took 1ul from the 1st PCR with 32 cycles, and input for the 2nd PCR which gave a nice band.
So any suggestions?