We would like to increase the yield of our PCR product. We are running a series of PCR reactions that is targeting ~1.1kb sequence. We begin each reaction with ~400pg of template DNA (total vector size ~6.1 kb), Fwd/Rev Primer concentration of 0.3uM (Tm of 62C and 60C, respectively), dNTP mix of 200um per nucleotide, 0.3mM MgCl2, and 1X buffer. We bring the total volume to 50 uL using DepC treated water.

Our cycle is as follows:

94C - 5 min

94C - 30 sec, 55C - 45 sec, 72C - 60 sec (Total of 30 cycles)

72C - 5 min

We use a NEB Monarch Spin Kit (loaded the 50 uL PCR product), followed the procedure, and eluted our DNA in 20uL of elution buffer and got a yield of 7.7 ng/uL. We need to have a yield of roughly 1 ug.

How do you suggest we can improve the yield of our PCR product?

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