Hello,
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?
1ug is only 7 tubes worth of purified material. Alternatively you could run a couple more cycles pre purification. If the product is a clean band with no obvious primer dimer you could exo-sap the pcr reaction to get rid of the primers and ntps to avoid losses on the purification column.
Have you tried a second elution off the purification column which might increase your yield
I think slightly increasing the number of cycles to 40 can significantly increase Your yeild
Just add up to 45-60 μL elution buffer. Also try 35 cycles.
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