How do I calculate the 15-nt overlap if the vector is linearized via restriction digest, generating a 5' or 3' overhang?
How do I calculate the 15-nt overlap if the vector is linearized via restriction digest, generating a 5' or 3' overhang?
The 5' overhang of a restriction site is included in the 15-nt complementary region. The 3' overhang of a restriction site is excluded from the 15-nt complementary region. Figures 2 and 3 in the In?Fusion Snap Assembly User Manual or In?Fusion Snap Assembly EcoDry User Manual provide detailed examples.
Restriction sites used for vector linearization can be preserved in the recombinant vector by adding nucleotides to the PCR primers between the template-specific portion and the 15-nt homologous overlap. The online Primer Design Tool allows you to choose whether or not to preserve the restriction sites. (The Primer Design Tool is compatible with Mozilla Firefox or Google Chrome web browsers, but not with Internet Explorer).