fusion PCR - loss of template

[majihec]

Hi,

I am performing a PCR fusion of 3 fragments. The total volume of reaction is 360 ul , and is divided into 12 tubes (30 ul each). Amount of templates in whole 360 ul: 400 ng (plasmid, size 4.0 kb), 350 ng and 350 ng (genomic DNA, size approx. 1000 bp each). The fusion product is then purified on column (Qiagen), digested and extrated from agarose gel (Qiagen kit). I have made several attempts, bud I haven't got any fusion product yet (size approx. 4 kb).

I tried to take reaction from 1 tube (30 ul) before and 1 tube after PCR and run agarose gel electrophoresis and I found that the amount of 400ng fragment decreased - at least by 1/2, maybe even by 2/3.
Corresponding fractions after purrification and digestion seems to be the same quantity as fraction after PCR.

Why is my plasmid template disappearing? Thanks a lot!

[jonoave]

In general, the amplification efficiency of plasmid is lower compared to double-stranded DNA, (not sure if I remember correctly) due to the circular double-stranded nature of plasmid or something.

I haven't done a lot of fusion work, but perhaps you can try fusing 2 templates (e.g A+B, A+C) at one time, separately? Then only you do a final fusion, i.e. AB + AC.