Primer design for USER cloning

Primer design for USER cloning is divided in two, depending on the primer region:

Binding region

This is the primer region that anneals to the target DNA sequence. This sequence is designed following the standard rules for primer design in a standard PCR.

5' overhang region

This primer region is responsible for the formation of the 3' cohesive overhangs that allow the subsequent ligation of DNA fragments. There are three main rules to be taken into account when designing this region:

• Length: The ideal length of a primer overhang is ca. 7-12 nucleotides. Longer overhangs are also valid, but the longer the overhangs, the lower the PCR yield.

• Melting temperature (Tm): The ideal range for the overhangs is ca. 10-30 °C. The primer overhangs Tm will define the 3' overhangs Tm in the DNA fragments to assemble. Therefore, it is important that the Tm for all DNA fragments to assemble were very similar to ensure a successful USER cloning experiment.

• DNA sequence design: 5' overhangs need to be as much different as possible to ensure a directed assembly of all DNA fragments.