Hello,

I am trying to assemble 2 fragments into a vector. The first fragment size is 820 bps, second fragment is 141 bps and the vector is 5373 bps and all fragments and the vector have 25-30 bps overlap with one another. I ran the assembly last week with 25ng of vector (0.007 pmol), 21 ng of first fragment (0.04 pmol) and 10 ng of second fragment (0.1106 pmol) but it didn't work. I had colonies after transformation but when I selected about 6 colonies and sent for sequencing the result showed that none of the fragments have been inserted and the vector seemed like it has been self-ligated which does not make sense at all, since the two ends were not overlapping at all! Here is my question, in the protocol has been indicated that if inserts are less than 1Kbp use 5 molar excess and when I planned to use 5 molar excess the amount of 141 bp fragment is too low (4 ng) which is not within the limit (10-20) so I did for 10 ng and it became like 15.8 molar excess compared with vector and 2.5 molar excess compared with 820 bp fragment. Should I just have both inserted in an equimolar amount?