Hi, recently I am trying to build up a DNA library derived from circulating DNA.

I used two-step strategy:

1) cell free DNA were phosphorylated using T4 Polynucleotide Kinase (first phosphorylation);

2) 5-fold of adaptors (unphosphorylated) were ligated with phosphorylated DNA using T4 DNA Ligase (first ligation);

3) Products from step 2 were purified using magnetic beads to remove the free adaptors;

4) After DNA purification, DNA was again phosphorylated using T4 Polynucleotide Kinase (second phosphorylation);

5) After second phosphorylation, the products were further treated with T4 DNA Ligase for nick ligation. Here, we can obtain a cell free DNA library.

As proof-of-concept experiment, I used 60bp blunt end dsDNA and 20 bp adaptor.After the two-step strategy, I run a PCR using universal primers (targeting adaptors), followed by agrose electrophoresis validation. However, the products were all about 40 bp that may be largely from adaptor self-dimerization.

Can anyone help me please? Thank you so much!