Hello,

I tried to clone 3 insert into plasmid pUC18. I used Takara infusion cloning with this reaction:

1 μl 5X In-Fusion HD Enzyme Premix

0.59 μl Linearized vector (2667bp, DNA mass=44.35 ng, conc=179.1 ng/ul)

0.85 μl Purified PCR fragment I (3740 bp, DNA mass=62.19 ng, conc=175.4 ng/ul)

0.77 μl Purified PCR fragment II (482 bp, DNA mass=16.03 ng, conc=99.2 ng/ul)

1.84 μl Purified PCR fragment III (1694 bp, DNA mass=28.17 ng, conc=36.6 ng/ul)

In total I have 5ul reaction volume and do the infusion reaction for 20 minutes in 50 degree C.

I used NEB calculator to get my DNA mass. Is my calculation correct. I search in takara website they have their own calculator to count the DNA mass. Please give me suggestion is there something wrong with my method? I have been failed working with this clone for 3 months.

Thanks